Biology 311 Human Genetics
Take-Home Exam I (80 pts.)
Due in class Friday Oct. 27, 2006
On separate pages, briefly TYPE DOUBLE-SPACED your answers to the following questions.
Copies of the papers for question 5 will be available as downloadable PDF files on Blackboard.
Feel free to consult the instructor if you need clarification on the questions. It is highly likely that she is the only one on the planet that has any idea where some of these questions are coming from.
I expect this take-home exam to be your own work. You are allowed to use student presentations on methods for information (some are posted on Blackboard), but make sure the information is correct and cite the particular presentation you used as a reference.
If you use any other references besides your textbook, be sure to cite them by author, year, title of book or journal, volume, and pages or provide the URL address for web sources. Exams that do not reference sources will be penalized.
1. (8 pts.) a. What is the most likely mode of inheritance of the trait shown in the pedigree below (autosomal dominant or recessive, X-linked dominant or recessive, or Y-linked)?
b. What is the genotype of individual III-1 in the pedigree? Define the allele symbols you use.
c. What evidence can you provide against other modes of inheritance? Hint: You may want to duplicate the pedigree and work through all the possibilities to identify individuals inconsistent with that particular hypothesis.
2. (6pts.) a. What is a dot blot?
b. Under what conditions would a short (20 nt) oligonucleotide bind to a specific DNA sequence in a dot blot? What would you change in the hybridization conditions to enable a similar oligonucleotide containing a single base pair mismatch to bind?
c. A dot blot DNA test involving allele-specific oligonucleotides (ASOs) was developed for a human autosomal recessive disorder.
+ASOoligo to normal allele
mASOoligo to mutant allele
You perform a dot-blot hybridization on DNA isolated from a patient who wants to learn if they are a carrier for the disorder. The test results as compared to those from individuals with various genotypes are shown below. The test results as compared to individuals with various genotypes are shown below; filled circles=hybridization, open circles=no hybridization.
Patient / +/+ / +/m / m/mDot blot probed with +ASO / / / /
Dot blot probed with mASO / / / /
Is the patient a carrier for this recessive disorder? Explain based on the hybridization patterns shown above.
3. (24 pts.) For any three of the following, outline a sequence of experiments to
a. produce a human protein in bacteria
b. separate large chromosomal regions
c. identify a gene that is expressed in particular cancer cells, but not in normal cells.
d. map the transcription start site of a human gene
e. locate the position of a gene or marker on a particular human chromosome
f. determine the subcellular localization of a human protein
g. identify proteins that interact with each other
h. isolate the 5’ end of a human cDNA
Be sure to include necessary steps and controls; detailed procedures can be abbreviated or referenced.
4. (12 pts.) Define six of the followingseven terms and indicate how they are relevant to a particular molecular genetics method.
a. yeast artificial chromosome (YAC)
b. DNA chip
c. DNA polymerase I Klenow subunit
d. oligo dT cellulose column
e. fluorophore
f. biotin-streptavidin
g. M13 phage
5. (30 pts.) Before he was selected to head up the Human Genome Project, Francis Collins (along with Lap-Chee Tsui) developed new methods that led to the discovery of the gene responsible for cystic fibrosis. Their general strategy of isolating a gene first, then figuring out what it does is called "reverse genetics". Further studies in other labs have helped explain why a deleterious allele for CF is maintained in the gene pool. You may wish to consult any or all of the following papers to answer the questions below--be sure to cite which ones you use. These papers can be downloaded from Blackboard or from the Milne library web site (although this can be tricky and requires patience). There also many more general references on the web for CF you may wish to consult.
- Rommens, J. M. et al. 1989. Identification of the Cystic Fibrosis Gene: Chromosome Walking and Jumping. Science 245, 1059-1065.
- Riordan, J. R. et al. 1989. Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNA. Science 245, 1066-1073.
- Pier, G. B. et al. 1998. Salmonella typhi uses CFTR to enter intestinal epithelial cells. Nature 393, 79-82.
a. Describe the nature of cystic fibrosis, its mode of inheritance, and illustrate its transmission in a typical pedigree.
b. What chromosome is the CF gene located on? Describe two different approaches that could be used to map a gene to a particular chromosome.
c. What is a genomic library? Why were genomic libraries essential to isolation of the CF gene? Describe how a lambda phage or cosmid genomic library, such as those used in these studies might be constructed.
d. What is chromosome walking? How does it differ from the new method Collins developed, called chromosome jumping?
e. The regions of the CF gene that code for protein were identified by comparing a series of overlapping cDNAs with the genomic region. What are cDNAs and how are they synthesized? How was the amino acid sequence of the CF protein (CFTR) deduced?
f. Describe the method used to identify the most commonly observed mutation seen for CF (ΔF508; shown in Fig. 4 of Riordan et al. 2004).
g. Heterozygote superiority is a possible hypothesis for the maintenance of the mutant CF allele in the population. Devise an explanation of how the typhoid bacterium might contribute to maintenance of CF heterozygotes. Describe some of the experiments used by Pier et al. to test this hypothesis and the results they obtained.
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