Practice Problems 8. a) What do we define as a beneficial or advantageous mutation to the virus? Why?

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1 Life Sciences 1a Practice Problems 8 1. You have two strains of HIV one is a wild type strain of HIV and the second has acquired a mutation in the gene encoding the protease. This mutation has a dual effect in that it causes a decrease in protease enzyme activity of 100 fold compared to wild type virus, and it also affects the binding of the protease inhibitor ritonavir so that ritonavir is 500 fold less likely to bind. a) What do we define as a beneficial or advantageous mutation to the virus? Why? b) If this mutation arose in an untreated HIV infected individual would the mutation be deleterious, beneficial, or neutral with respect to the virus? Would the mutation be retained in fifty generations? Briefly explain. c) If this mutation arose in an HIV infected individual taking the protease inhibitor ritonavir would the mutation be deleterious, beneficial, or neutral with respect to the virus? Would the mutation be retained in fifty generations? Briefly explain. 1

2 2. You decide to design an experiment so you can witness evolution in action. You choose bacteria for your experiment since they divide every twenty minutes. You will treat the bacteria with vancomycin, an antibiotic that inhibits the synthesis of bacterial cell walls, and see if the bacteria develop resistance to vancomycin. First you pick a single colony of wild type bacteria from a plate and grow the bacteria in a culture (Culture A) containing 10 milliliters of media overnight. You also pick a second colony of wild type bacteria and grow this bacterial colony in 10 milliliters of media containing vancomycin overnight (Culture B). In the morning, you take 10 microliters (1/1000 th ) of each culture and spread the 10 µl sample on a plate with media and a second 10 µl sample on a plate with media plus vancomycin. You incubate the plate overnight and count the colonies the next day. Bacterial Colony grown in 10 ml culture #colonies on minimal media plate #colonies on minimal media plate + vancomycin Culture A: Media Culture B: Media +Vancomycin a) At the start of the experiment both bacterial cultures A and B had the same number of bacteria. Based on the number of colonies on the minimal media plate how many bacteria were in the original cultures at the end of the incubation period? b) Why is the number of bacteria in the two cultures different at the end of the incubation? c) Why did culture B grow more colonies (200) on the plate containing media + vancomycin than culture A (1 colony)? d) Would mutations occur with similar frequencies in the two cultures A and B? 2

3 You next take the single colony from culture A (media only) that grew on the plate + vancomycin. You put it in two new 10 ml cultures one containing media (Culture C) and one containing media + vancomycin (Culture D) and grow the bacteria overnight and then replate samples from both cultures on plates. Bacterial Colony from A grown in 10 ml culture #colonies on minimal media plate #colonies on minimal media plate + vancomycin Culture C: Media Culture D: Media +Vancomycin e) Why are there fewer colonies on the plate containing vancomycin from Culture C than from Culture D? f) Why do more bacterial colonies from Culture C grow on the minimal media plate (950) than on the plate containing vancomycin (100)? What does this tell you about resistance to vancomycin? 3

4 3. Ribozymes are RNA molecules that act as enzymes to catalyze chemical reactions. Many natural ribozymes exist that catalyze their own cleavage or that of other RNAs. Scientists set out to determine if additional ribozymes could be generated and evolved from random RNAs. Approximately random synthetically generated nucleotide sequences were tested for their ability to cleave an RNA substrate. If a sample showed catalytic activity (defined as cleaving at least 1 RNA substrate molecule every 1000 minutes), that sequence was re-amplified in an error-prone (mutation introducing) process and re-tested. 15 rounds of screening and amplification were performed to eventually generate 12 different classes of RNA cleaving ribozymes. Ribozymes belonging to these 12 classes enhanced the rate of RNA cleavage 10 3 or 10 6 fold over the uncatalyzed rate of RNA cleavage. Moreover of these 12 classes, only one of them corresponded to a naturally occurring ribozyme (Class IX shown below). (J. Tang and R. Breaker PNAS. Vol. 97, no. 11.) a) In the above figure, the arrow represents the location where the RNA gets cleaved. It has been observed that these cleavage sites are almost always outside of helical regions (nonbase paired regions). Based on your knowledge of the stability and structure of nucleic acids, why would non-base-paired sites be more accessible for cleavage? 4

5 b) It was observed that with each round of error-prone replication, more ribozymes were generated. How could the introduction of more errors lead to the generation of new ribozymes? c) Out of tested RNAs, approximately 100 different ribozymes with distinct sequences were isolated. What is the frequency of finding a ribozyme when testing random nucleotide sequences? d) In this experiment, what was the selective pressure used to produce these ribozymes? Was it naturally occurring, or artificial (man-made)? e) The rate of catalysis by these newly discovered ribozymes is times slower than that of naturally occurring ribozymes. How could the scientist generate ribozymes with greater catalytic activities? 5

6 4. Retinoblastoma (Rb) is a childhood cancer that originates in immature retinal cells in one or both eyes and can occur in the womb up to 5 years of age. Rb occurs when a cell loses the function of the protein encoded by the RB1 gene. The occurrence of Rb is approximately once every 20,000 births. 90% of the cases have no family history of the disease, however 10% of the cases do have a family history. a) Most people have two functional copies of the RB1 gene. Where do we get these two copies of RB1? b) RB1 encodes a protein that regulates the proliferation of cells. Is there a difference between cell proliferation and cell growth? c) When RB1 function is lost, cells proliferate rapidly. What does this suggest is the function of the RB1 protein? d) In patients without a familial history, usually only one tumor arises in one eye. However in patients with a familial history, multiple tumors arise and often in both eyes. Based on your understanding of the origins of tumors, why do patients with a familial history develop multiple Rb tumors in both eyes? e) Would a Rb patient with a familial history of Rb have a greater risk of having children with Rb than a Rb patient without a familial history? Why? 6

7 f) Which of the following would have a greater risk of having children with Rb: a person who has never had Rb, or a person who has non-familial Rb? Why? 5. The number of newly diagnosed cases of colon cancer in women in England and Wales in one year is plotted below as a function of age at diagnosis and expressed relative to the total number of individuals in each age group. The incidence of cancer rises steeply as a function of age. (Data from C. Muir et al., Cancer Incidence in Five Continents, Vol. V. Lyon: International Agency for Research on Cancer, 1987.) a) Assume mutations occur with a similar frequency throughout a person s lifetime. If a single mutation were required to trigger the development of cancer would the curve look like the one above? If not how would the curve change? Please draw one possibility. b) How many mutated cells does a tumor derive from? 7

8 c) Multiple factors can increase the risk that an individual will develop cancer during their lifetime. Name two general categories. d) Cancer is often more treatable the earlier a tumor is detected. Recently some clinics have advocated that healthy patients have yearly whole-body CT scans to detect tumors prior to the presentation of any clinical symptoms. Unfortunately these scans expose the patient to an extremely high dose of X-rays, which can cause DNA damage. It has been estimated that having annual scans from the time a person is 35 to 70 years old increases the risk of cancer by 2%. Under what circumstances would a whole-body scan be a good idea? e) If ten years from now, a 10% increase in cancer diagnoses is observed in patients who have had annual full-body scans, is it possible that the scans contributed to the development of the disease? If so, why was there a lag in time? f) Assuming that it could be determined that a whole-body scan was the cause of a patient s tumor, which of the factors listed in c would a whole-body scan fit within? 8

9 1. HIV and protease mutations a) A change in the virus genetic material that allows this mutated virus to produce more viable progeny in every generation. This virus will out compete other strains of virus and eventually take over the population. b) In an untreated HIV infected individual this mutation would be deleterious for the virus since this virus would have a protease with 100 fold lower activity. This virus would produce progeny more slowly and would be out competed and lost. c) In an HIV patient taking ritonavir this mutation would be beneficial with respect to the virus. The virus would be much less affected by the protease inhibitor ritonavir. The advantage gained by less inhibition would be greater than the disadvantage resulting from the lower protease activity. This virus would be retained in fifty generations. 2. Bacterial experiment a) # bacteria in culture= number on plate X dilution factor Culture A: 1000 X 1000 = 1,000,000 bacteria in 10 ml culture Culture B: 205 X 1000 = 205,000 bacteria in 10 ml culture b) In order for the bacteria to survive and divide in culture B they had to mutate and develop resistance to vancomycin. Only a portion of the population was able to develop beneficial mutations and survive, those that did not died. Therefore there are fewer bacteria in the vancomycin containing culture at the end of the incubation than in the culture of bacteria with media alone. c) The bacteria in culture B had been exposed to vancomycin since the culture was started. To survive in culture B, a bacterium needed to develop resistance and then it could divide. Virtually all of the bacteria in culture B should be vancomycin resistant. In liquid culture A, a bacterium that developed resistance to vancomycin would not be selected for, although a spontaneous mutant could arise (for instance the one surviving plated colony). d) Yes, but the mutation would only be selected for and take over the population in culture B. e) Since bacteria from culture C were not continually selected with vancomycin, most bacteria lost their resistance to vancomycin. In culture D the bacteria continued to grow in the presence of vancomycin so they needed to retain resistance to vancomycin in order to survive. f) The majority of bacteria in culture C have lost their resistance to vancomycin. This implies that the development of resistance to vancomycin affects the growth rate of bacteria; resistance to vancomycin causes a loss in rate of 9

10 growth. [Acceptable alternative: a continuous exposure to vancomycin is required for the mutation that causes resistance to be retained.] 3. Ribozymes a) Non-based pair sites would be more accessible to cleavage with water since they would not form a Watson-Crick double helix shielded by two negatively charged phosphate containing backbones. b) The introduction of more errors caused the ribozymes to acquire more functions. c) 100/ = 1 in d) The selective pressure was the test of the ribozyme s ability to cleave the RNA substrate. It was artificial selection; ribozyme s were selected for catalytic activity based on criteria set by the scientists performing the study. e) They could be more selective and require a higher catalytic activity to be classified as a ribozyme (define activity as cleaving at least 1 RNA substrate molecule every 100 minutes-instead of 1 every 1000 minutes) or They could optimize the conditions to generate more ribozymes. 4. Retinoblastoma (Rb) a) One copy comes from each parent. b) Yes. Cell proliferation refers to an increase in cell number and cell growth refers to an increase in the mass of a cell. c) This suggests that the RB1 protein is involved in blocking or slowing cell proliferation. d) Patients with a familial history have inherited one mutant (non-functional) copy of the RB1 gene. There chances of acquiring a mutation in the second copy of the gene are fairly high resulting in multiple tumors. Patients without a familial history have two wild-type copies of the gene, for a tumor to arise they have to develop mutations in both copies of the gene. This is a fairly rare occurrence so they usually only develop a single tumor (recall the dice analogy). e) Yes, since the Rb patient with a familial history inherited the mutant copy of the RB1 gene. The mutant RB1 gene is present in all of their cells, including their germ cells. They will have a 50% chance of passing the mutant gene to their children. The Rb patient without a familial history likely has a mutation that arose spontaneously in one of their somatic cells, in this case they would not pass the mutated RB1 gene to their children. 10

11 f) They would have the same risk of having children with Rb. The individual with non-familial Rb developed a mutation in a somatic cell, this mutation would not be passed on to future generations. Alternative answer: A person who has non-familial Rb would have a greater risk of having children with Rb. Since they spontaneously developed a mutation in RB1 they might have some low genetic propensity for the development of Rb. 5. Incidence of colon cancer a) No, it would not look like the curve above. It would be a flat line such as the black line shown above. b) One c) Environmental Genetic d) It would be a good idea if the patient is at a high risk of developing a cancer that could be detected by a whole-body scan. Otherwise, it would not be advisable to have yearly whole-body scans. e) Yes, it is possible. The lag in time would come from the fact that for the patients to develop cancer multiple mutations needed to occur in addition to the mutations caused by the full-body scan. f) Environmental 11