7.014 Problem Set 7 Solutions Question 1 Part A Light chainLight chainHeavy chainVariable regionVariable regionAntigen binding siteAntigen binding siteMembrane-spanning region of a membrane-bound antibodyRegion of secreted antibody that interacts with phagocytes Heavy chainMembrane-spanning region of a membrane-bound antibody On the diagram label the following structures (a) – (d) on the antibody diagram. a) the antigen binding sites b) the heavy chains and the light chains c) the variable regions d) i) If this was a membrane-bound antibody, indicate the membrane-spanning region of the antibody ii) If this was a secreted antibody, indicate the region of the antibody that would interact with phagocytic cells e) List two ways that combinatorial joining generates antibody diversity. 1) Association of different light chains and different heavy chains 2) Recombination of different V (D) and J segments on the immunoglobulin genes. also: Imprecise joining of nucleotides at the joining sites.2 Question 1, continued Below is a schematic of the unrearranged immunoglobin heavy chain locus. V segments D segmentsJ segmentsconstant segmentregion aregion bregion c region dregions e a) Indicate which region(s) contribute(s) to the formation of the antigen binding site of the antibody molecule. Regions a, b, c b) After rearrangement, which region(s) would be the same in different B cells? Region d c) Which region(s) would never appear in any mRNA transcribed from this locus in a population of mature B cells? Regions e d) Cindy wants to store cell samples from her chinchilla so that in the future she can produce a clone. She has isolated cell lines from the following four different cell types. However, freezer space in the lab is limited and she can keep only one. Which cell line should she keep? Red blood cell Gut cell B cell T cell Why keep this cell line rather than the others? She should keep the gut cell line. Red blood cells do not have nuclei, and B and T cells have rearranged the DNA at the antibody and T cell receptor loci, respectively3 Question 1, continued Part B The virus which causes AIDS (acquired immune deficiency syndrome) is called HIV (human immunodeficiency virus). HIV is a retrovirus, and its genome is a single RNA strand. Two copies of this RNA genome are packaged with two copies of the retroviral enzyme, reverse transcriptase, within a protein capsid. a) For HIV infection to occur, a surface protein on the virus must interact with a surface protein on the cell. HIV specifically infects the helper T cells of the human immune system. Give one reason that HIV infection is specific for helper T cells. A viral protein on the surface of the HIV particle binds to a protein that is only found on helper T cells. b) Once infection has taken place, the RNA genome has to be made into double-stranded DNA. This process is mediated by reverse transcriptase. Once a double-stranded DNA copy of the HIV genome has been made, it is integrated into the host cell genome. The integration event is mediated by an enzyme, integrase. i) What are the three steps required to produce double-stranded DNA from the single strand RNA genome? 1. Synthesis of the complementary strand of DNA using the single strand RNA genome as the template. 2. Degradation of the single (+) strand RNA template. 3. Synthesis of the complementary strand of DNA using the single strand of DNA as the template to form double-stranded DNA. ii) The central dogma states that: DNA RNA Protein Which one of the enzymatic activities possessed by reverse transcriptase goes against the central dogma? Explain your answer. The RNA-directed DNA polymerase activity used to synthesize a single strand of DNA from a single strand RNA template. The central dogma states that only DNA can act as the template for DNA synthesis (in replication), and that only DNA acts as the template for RNA synthesis (in transcription). The drug most widely used to combat AIDS is Azidothymidine (AZT). AZT is very similar to thymidine, except that the 3' hydroxyl (OH) group on the deoxyribose ring has been replaced by an azido (N3) group. c) Which process of the life cycle of the HIV do you think is inhibited by AZT? As an analog of thymidine, AZT interferse with the synthesis of DNA. HIV reverse transcriptase incorporates AZT instead of thymidine into the growing DNA and it blocks further chain elongation because there is no 3' OH group. d) What side effects, if any, do you expect from AZT treatment? While AZT will inhibit DNA synthesis mediated by reverse transcriptase, it will also inhibit DNA synthesis in other cells and thus kill cells that are actively dividing (e.g. red blood cell precursors, skin cells, etc. However, HIV reverse transcriptase prefers to use AZT over thymidine while host cell DNA polymerase prefers thymidine over AZT. Therefore, AZT may be used at concentrations that inhibit HIV replication but are not yet toxic to host cell polymerases. e) AZT-resistant forms of HIV have been isolated. These mutant viruses have mutations in one of the viral genes. In which viral gene do you think are the mutations most likely to be found? Why? The mutations are most likely to be in the viral gene coding for reverse transcriptase (the pol gene) because mutations in this gene may directly cause the mutated reverse transcriptase not to prefer AZT during replication and thus become resistant to AZT.4 Question 2 a) Below is a Cohort Life Table for a population of gray squirrels. Fill in the remaining spaces: Gray Squirrel Cohort Life Table x nx lx dx mx Lx Tx ex bx lxbx 0 500 1 0.7 0.7 325 794 1.588 0 0 1 *150 0.3 0.06 0.2 135 469 3.127 0.5 0.15 2 120 *0.24 0.048 0.2 108 334 2.783 2 *0.48 3 96 0.192 0.038 0.2 *86.5 *226 *2.354 2 0.348 4 77 *0.154 *0.03 *0.2 69.5 139.5 1.811 2 0.308 5 62 0.124 *0.062 *0.5 46.5 70 1.129 *1 0.124 6 31 0.062 0.046 0.75 *19.5 *23.5 *0.758 0 0 7 8 0.016 0.016 1 4 4 0.5 0 0 8 0 0 0 0 0 0 0 0 0 b) When the table was made, some of the values were measured and others were calculated from those values. Which three values in the table must be measured in order to construct the rest of the table? x, nx, bx The gray squirrels have a high infant mortality rate (mx), but those that survive tend to not die of natural causes until age five to seven. In the meanwhile, those
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