LB-145 Exam II has two parts (Part 1 is worth 30 points)

Part 1 of your exam is open-book, open-library, open-internet and you are

encouraged to discuss these questions with members of your lab group. *But* write your answers on your own, in your own words, as described below.

• You have until midnight Monday March 18th to compose answers to all six

questions listed. You will turn in your answers by uploading them to turnitin.com by midnight (11:59pm) Monday night & then submitting a hard copy by 3pm at the start of the lecture meeting the next day, Tuesday March 19th.

• Your answer to each of these six (6) questions cannot be more than 300 words of

text, and your text and hand-drawn illustrations combined cannot take up more than both sides of a single (1 answer/1 page) sheet of paper for each question.

• The text of your answer must be submitted to http://turnitin.com/ by the deadline,

yet since this is an exam, only submit your final answers, not drafts, and be aware you will not be able to view the “similarity report.”

• We will randomly chose one answer and grade only it (30 points).

• On Thursday March 21st you’ll then take Part 2, an in-class, individual and

closed-book test. Part 2 of the exam will have 2-3 essay questions that will be the same as, or comprised of elements from, just these six questions (70 points).

“Individual Responsibility” Signature Page (Exam II) Print out this sheet to serve as the front cover of exam II.

I, the undersigned, am verifying that as a member of the Lyman Briggs community, I have held myself and my peers to the highest measures of honesty and integrity. My written answers are my original work and I have neither given my written answers to others or received written answers from others. I used no images or text from other students or from published resources in print, internet or other domains in the composition of these essay answers; these words and illustrations are my own. I have submitted this work to turnitin.com. If I plagiarize I understand I will be given a zero for the exam and course. Name (type initials and B-PID to serve as signature but maintain "blind grading")

Initials:__________ B-PID: ____________

Questions: Answer the six questions below with full sentences and explanations (and illustrations if required, or if you deem them helpful). To earn more points, include footnote citations in your answers to identify when your ideas are supported by sources. To provide more support for obtaining full credit for an answer, predict more than just one correct solution if possible.

1. Predict how many photons of red light are minimally required to create one glucose molecule in photosynthesis (or two glyceraldehyde-3-phosphate molecules) and explain your reasoning. (i) Illustrate the non-cyclic electron transport version of light reactions and the Calvin cycle. (ii) Explain in full sentences your prediction and your rationale for it. Explain in detail the sequence of events that occur in the process of photosynthesis and your step-by-step rationale/logic in your calculations [Assume: the Calvin cycle must occur in full cycles, you must only use linear electron transport, 3 H+ travel through ATP Synthase for it to generate one ATP]. No limit to illustration but must fit in 8x11 page. Label well, but judiciously. Text limit is no more than 300 words.

2. Draw an epithelial cell and in the illustration communicate all the steps necessary to create a CFTR protein through biosynthesis (start at gene on chromosome; end at final destination, draw and list all possible events that occur at each location along the way). No limit to illustration but must fit in 8x11 page. Label well, but judiciously. Text limit is 300 words in total.

3. Sickle cell disease was the first time anyone found the molecular cause for a genetic disease.

a) What causes the red blood cells to change their shape? Support your answer with data and limit your answer to a maximum of four sentences.

b) What is the specific difference between wild-type and the sickle cell protein? Support your answer with data and limit your answer to a maximum of four sentences.

c) Does this bear any similarity to cystic fibrosis? Support your answer with sources, and limit your answer to a maximum of four sentences.

d) Why has sickle cell disease persisted, given how natural selection works (shouldn’t that allele leave the gene pool over time)? Support your answer and limit it to four sentences.

4. What parameters cause DNA polymerases to generate more mutations? What are the health implications for the data in Tables 5.1, 5.2, 5.3 of your textbook with regards to cancer, aging and exposure to metal ions? Claim: Evidence: Reasoning: 5. Explain how the experiment in Figure X was a clinical failure but a basic research success.

You may be surprised to learn that the information content of DNA can be modified chemically without altering its sequence of base pairs, similar to capitalizing some letters without changing the words: tHE LoveLy Oven. If you read all the letters equally, you get an opinion about a kitchen appliance. However, if you read only the upper case letters, you get a salutation. To read “HELLO,” each letter’s case matters, and in DNA, chemical modification of nucleotides matters. For example, if chemical modifications can alter the information carried by DNA, then perhaps modified DNA could help explain some illnesses that are caused by environmental factors such as cigarette smoke and high fat diets. Once biologists realized there was more to DNA that just the order of base pairs, investigators wanted to understand the full extent of the epigenetic modifications and their consequences.

Sickle cell disease is caused by a gene that encodes for a malfunctioning protein. The aberrant protein is encoded by a hemoglobin gene. All primates, including humans, have several hemoglobin genes that encode the proteins responsible for carrying oxygen in our red blood cells. Sickle cell disease hemoglobin causes red blood cells to become deformed and sometimes resemble a sickle, or a crescent moon. The abnormally-shaped red blood cells clog the smallest capillaries, leading to many circulatory problems and tissue damage. The molecular information that causes sickle cell is in the DNA sequence of every cell in the patient’s body. The investigators wanted to block the disease symptoms by turning on an alternative hemoglobin gene that was not mutated but normally silent in adults. Their idea was to use epigenetic manipulation of a methylated hemoglobin gene and replace the faulty hemoglobin protein with a properly functioning hemoglobin.

Figure X Monkey response to 5-azaC injections. Fetal hemoglobin levels were measured after monkeys were injected with 5-azaC at four times (A-D). More 5-azaC was injected at C and D than at A and B.

When you were born, your red blood cells were filled with a fetal form of hemoglobin encoded by a different gene than adult hemoglobin. The investigators wondered whether it would be possible to chemically manipulate the methylation of hemoglobin genes in sickle cell patients to activate the normally silent fetal hemoglobin gene. Other investigators had already discovered that C- and G-rich DNA segments adjacent to the fetal hemoglobin gene became hypermethylated 6 months after birth, perhaps explaining how this gene could be active during fetal development and infancy but silenced after 6 months of age. The clinical investigators chose a drug called 5-azaC that was already shown to prevent formation of m5C. The compound 5-azaC is a modified version of the base cytosine, but it cannot be methylated at the number 5 carbon. Because conducting preliminary research on humans would be unethical, the investigators used a monkey model for sickle cell for their first experiments (Figure X). They injected monkeys with two different dosages of 5-azaC over a period of a couple months, and they measured the change in fetal hemoglobin in the monkeys’ blood. The hemoglobin response indicated that in monkeys, and probably humans, the fetal hemoglobin gene activity could be increased by reducing epigenetic methylation of DNA. Therefore, they had the first evidence that epigenetic alterations could be used to treat diseases.

A. Trifecta: Based on the reading, compose a Trifecta for Figure X (purpose, methods, findings) B. Explain how the experiment in Figure X might be considered a clinical failure but a basic research success. Claim: Evidence: Reasoning:

6. Design a diagnostic test for the mutation that causes sickle cell anemia. Sickle-cell anemia is caused by the mutation of a single base in the gene that codes for the beta globin polypeptide of hemoglobin. Using Figure 1 and sequence information provided below, design a PCR-based test to diagnose the presence of mutation E6V in the human beta globin (HBB) gene.

Figure 1: Sickle-cell anemia is caused by the mutation of a single base in the gene that codes for the beta globin polypeptide subunit of hemoglobin. The change in amino acid sequence causes hemoglobin molecules to crystallize when oxygen levels in the blood are low. This causes red blood cells to "sickle". Note: During the normal post-translational modifications of beta globin the first amino acid, methionine, is always removed from HBB protein and thus valine then become the first one listed above. In the DNA sequences shown, the sense strand is oriented 5’-3’ but is located below the complementary antisense strand. a. Design primers [show exact sequences], predict annealing temperatures, and explain the temperatures and durations you’ll use for PCR in the thermocycler. b. Indicate you expected band(s) size and draw a predicted gel with the 1kb+ MW ladder as reference beside your predicted band(s). c. Discuss controls you would like to use to be sure your assay works well, and design additional tests you will do to confirm the identity of your PCR band. Wild-type normal HBB gene sequence [use this sequence and just assume it has no introns]

1 ACATTTGCTT CTGACACAAC TGTGTTCACT AGCAACCTCA AACAGACACC ATGGTGCACC 61 TGACTCCTGA GGAGAAGTCT GCCGTTACTG CCCTGTGGGG CAAGGTGAAC GTGGATGAAG 121 TTGGTGGTGA GGCCCTGGGC AGGCTGCTGG TGGTCTACCC TTGGACCCAG AGGTTCTTTG 181 AGTCCTTTGG GGATCTGTCC ACTCCTGATG CTGTTATGGG CAACCCTAAG GTGAAGGCTC 241 ATGGCAAGAA AGTGCTCGGT GCCTTTAGTG ATGGCCTGGC TCACCTGGAC AACCTCAAGG 301 GCACCTTTGC CACACTGAGT GAGCTGCACT GTGACAAGCT GCACGTGGAT CCTGAGAACT 361 TCAGGCTCCT GGGCAACGTG CTGGTCTGTG TGCTGGCCCA TCACTTTGGC AAAGAATTCA 421 CCCCACCAGT GCAGGCTGCC TATCAGAAAG TGGTGGCTGG TGTGGCTAAT GCCCTGGCCC 481 ACAAGTATCA CTAAGCTCGC TTTCTTGCTG TCCAATTTCT ATTAAAGGTT CCTTTGTTCC 541 CTAAGTCCAA CTACTAAACT GGGGGATATT ATGAAGGGCC TTGAGCATCT GGATTCTGCC 601 TAATAAAAAA CATTTATTTT CATTGCAA