Copy of Exam 1 Spring 2013

AVERAGE = 77.5% Principles of Cell Biology Exam 1 March 5, 2013 Directions: There are 75 questions in this exam with only one correct answer for each. All answers are to be placed on the grid sheet. Make sure that you mark the version of your exam for question #76. The version is listed at the end of this exam. Questions 1 through 50 relate to the following problem : You have a culture of human cells growing in a plastic cell culture plate in the presence of serum supplemented media and you are interested in what effects, if any, the hormone insulin might have on the change in the behavior of the cells. 1. You are interested in analyzing and tracking the insulin molecule and its receptor binding. To this end you label both the insulin molecule as well as the insulin receptor with CFP (cyan fluorescence protein) and YFP (yellow fluorescent protein), respectively. What technique are you performing in this case? a. FRET b. FACS c. FISH d. ELISA e. FRAP 2. To achieve an overall microscopic image of the cells you decide to fix the cells in formaldehyde, infiltrate them with paraffin, and subsequent to sectioning and processing you stain them with hematoxylin and eosin. In this context which microscope below would be the best to select? a. Atomic force microscope b. Two photon microscope c. Scanning tunneling microscope d. Bright field microscope e. Darkfield microscope 3. In another experiment you decide to measure the amount of protein secreted by these cells in response to insulin. You have a monoclonal antibody that is specific to the protein that you would like to measure. Of the following techniques which would be the best one to chose? a. Fluorescence immunocytochemistry b. Ultrastructural immunocytochemistry c. ELISA d. Electron tomography e. DEAE ion exchange chromatography

4. You now plan to analyze these cells using a spinning disk confocal microscope. You decide to select this microscope rather than the laser scanning confocal microscope. What is the basis for selecting the former over the latter? a. You can’t use the latter with fluorescent dyes b. Only the former can be used at NIH given that the spinning disk microscope is not yet commercially available c. You can only use plastic thin sections with the latter microscope d. The spinning disk is especially suited for analyzing dynamic events in living cells that occur during short periods of time e. None of the above 5. You notice that when you add large amounts of insulin many of the cells change their shape and morphology suggesting that large amounts of insulin may be killing the cells. To this end you decide to use Calcein-AM and Propidium Iodide as a live/dead assay to determine if this is the case. Which instrument below would be the best match for analyzing these probes? a. Plate reading spectrofluorometer/Cytofluor b. Velocity sedimentation c. Differential centrifugation d. Laser capture microdissection microscopy e. ELISA 6. You next perform a band/gel shift assay. What type of question are you asking? a. How does insulin interact with its receptor in the plasma membrane? b. Where in the cell are mitochondria located? c. What select proteins in the cell are binding to and possibly regulating specific genes? d. Can the cells in question synthesize collagen in response to insulin? e. Can the cells in question differentiate into fat cells? 7. One critical question in your experimental series is to determine if insulin increases the expression of a select mRNA and you would like to use a fluorescence microscope and/or a confocal microscope to determine where within the cell this mRNA is located. Which of the following would be best suited to address this question? a. FACS b. FRAP c. FRET d. FISH e. Only Dave Matthews knows for sure 8. Assuming that these are human kidney cells, you would like to change the substrate such that they might be coaxed into expressing their kidney phenotype better than if they were cultured on standard cell culture dishes. Based on the discussion in class, which of the following substrates would be the most logical to chose in this regard? a. Microporous membrane cell culture inserts b. Roller bottles c. Tubes d. Beads e. None of the above would probably encourage a more kidney-like phenotype

15. Dynabeads are available to use for your experiments. Which type of technique would best match Dynabeads? a. Confocal microscopy b. Analyzing SNPs c. cDNA microarrays d. Separating cells/organelles from each other e. Introducing shRNA into the cells 16. Rather than simply adding insulin to the media you now plan to microinject the hormone using a glass micropipette. Of the following microscopes below which would be most suitable for this purpose? a. Phase contrast microscope b. Nomarski/DIC microscope c. Scanning electron microscope d. Atomic force microscope e. 2 photon microscope 17. You find that using standard confocal microscopy imaging a fluorescent probe causes the cells to die through necrosis due to phototoxicity given that you are trying to image through several layers of cells. As such, you decide to choose another microscope that results in less phototoxicity when imaging many cell layers. Which of the following would be best for this purpose? a. Laser capture microdissection microscopy b. Two photon microscopy c. MRI microscopy d. Scanning tunneling microscopy e. Atomic force microscopy 18. You intend to expand the cultures over several days to generate a large number of cells. Which microscope below that relies on differences of refractive indices in cells would allow you to follow these cultures noninvasively given that you don’t have to fix or stain them? a. Phase microscopy b. Transmission electron microscopy c. Scanning electron microscopy d. Bright field microscopy e. None of the above would work in this application 19. One set of experiments requires that you analyze 1000s of mRNAs at once so that you can get a better sense of insulin-specific gene expression regulation. Which of the following non gel techniques below would allow you to do this? a. Western blots b. cDNA microarrays c. Southern blots d. Generating temperature sensitive mutants e. MALDI-TOF

20. You next do an experiment with western blots. Which of the following is typically not a key requirement for this type of analysis? a. Monoclonal antibody b. SDS gel c. Densitometer d. Blotting paper e. Centrifuge 21. You want to now determine if the interaction of insulin with these cells causes an increase in intracellular calcium. Of the fluorescent probes below, which would be the best choice? a. BCECF-AM b. Calcein-AM c. Propidium iodide d. Fluo-3 or Fura-2 e. Methylene blue 22. In another series of experiments you fix the cells with gluteraldehyde and osmium tetroxide following infiltration with plastic and cutting of ultrathin sections for analysis. What stains below would be most appropriate for use with this technique? a. Calcein-AM b. Fluorescein c. Lead and uranium d. Hematoxylin e. Eosin 23. You now plan for a tandem set of density gradient followed by rate zonal centrifugation experiments. What type of analysis are you planning on executing? a. Organelle separation b. phospholipid separation/analysis c. SNPs separation/analysis d. Isolation of monoclonal antibodies e. Protein X ray crystallography 24. You suspect that one of the proteins that i s triggered to be synthesized by insulin has an overall negative charge and you want to purify this protein based on this characteristic. Which technique below would make the best choice to select based on this characteristic of the protein to be separated? a. Gel filtration b. Density gradient centrifugation c. DEAE ion exchange chromatography d. CM ion exchange chromatography e. Electron tomography

30. Next you plan to add insulin and do a FRAP (fluorescence recovery after photobleaching) experiment. What particular parameter would you be most likely analyzing? a. Ability of the cells to differentiate b. Ability of the cells to divide c. Ability of two proteins to bind to each other within a 1-10nm distance d. Ability of a selected cell membrane molecule to diffuse laterally within the plane of the membrane e. None of the above 31. You now decide to do a SNP association study with this lot of human cells so that you can compare them to another lot of human cells procured from a different donor. What type of molecule are you most probably analyzing in this regard? a. Proteins b. DNA c. mRNA d. Carbohydrates e. Phospholipids 32. You next produce a monoclonal antibody that binds to a protein that you think may be increased in abundance when the cells are stimulated by insulin. Which of the following techniques available to you do not require the use of a monoclonal antibody? a. Southern blots b. Western blots c. Fluorescence immunocytochemistry d. ELISA e. Protein A immunoprecipitation 33. You now need to do a TIRF (total internal reflection) experiment and your colleague next door allows you to use his laboratory for this purpose. Which of the following best matches TIRF? a. Protein separation technique b. Microscope technique c. Cell culture technique d. Molecular biology technique e. Cell separation technique 34. You are interested in viewing the internal structure of the membrane of these cells relying on carbon-stablized, platinum replicas after their exposure to insulin so that you can visualize individual transmembrane proteins. Which of the following techniques would be best matched for this purpose? a. Plastic thin sectioning b. Paraffin sectioning c. Confocal microscopy with 3-D reconstruction d. X ray microscopy e. Freeze fracture

35. You next chose to use a Guava system to analyze your cells. What is required for this purpose? a. Colorimetric dyes b. Gold particles c. Liposomes d. Fluorochromes e. Radioactive probes 36. One of your experiments requires you to use 35 S-methionione to track insulin-specific protein synthesis. You harvest the cells and isolate the proteins using SDS gel electrophoresis. Which of the following steps would be required next to visualize the radioactively tagged bands? a. FACS b. FISH c. Autoradiography d. TIRF e. Two photon microscopy 37. You hope to make mutant cell lines from these human cells. Which of the following would be the best treatment on the path to generate these mutant cells? a. Ethylmethane sulfonate (EMS) b. Triton X 100 c. Speedbeads d. Sodium dodecyl sulfate (SDS) e. EGTA 38. One of your aims is to determine if insulin affects the length of telomeres as cells divide. What is the importance of telomeres in this regard? a. Telomeres are involved in the degradation of mitochondria b. Telomeres trigger cells to differentiate c. Telomeres are absent in all human cells except for human embryonic stem cells d. Telomeres are the “mitotic clock” of cells that can influence how many times cells divide. e. None of the above 39. You notice the human cultures you are working with have a heterogeneity that suggests that they may consist of several different types of cells given their different physical sizes. As such, you decide to perform a cell separation experiment that separates these cells based on size. Which of the following best matches how you would do this experiment? a. Velocity sedimentation b. Density gradient centrifugation c. FACS d. Laser capture microdissection microscopy e. None of the above are separation techniques that separate cells based on size

46. Your research associate approaches you with an idea to generate temperature sensitive mutant cell lines from these cells. You explain, however, that temperature sensitive mutants can’t be typically generated from mammalian cells. Rather, they can be generated using what type of cells/organisms that were critical in the 1970s to first identify and then understand the cell cycle genes? a. C. elegans b. S. aureus c. Yeast cells d. Mycoplasma e. Viruses 47. In a final set of experiments you chose northern blots as your technique. What is this technique designed to measure? a. Protein synthesis b. Carbohydrate turnover c. Degradation rate of proteins in the lysosomes d. Number of genes in the nuclear genome e. mRNA expression 48. You now hope to generate a “knock in” cell line from these human cells by integrating a select gene that you have previously characterized into the nuclear genome. This process is called a. Disposition b. Translation c. Transfection d. Trepidation e. None of the above 49. You identify a gene of interest in your cells and you now want to expand the amount of this gene experimentally so that you can use it for designing other types of techniques. Which of the following below would be the best choice in this regard? a. FRET b. FRAP c. PCR d. shRNA e. hESC 50. As your final experiment you now decide to do a chromatography experiment using insulin that is covalently attached to the bead with the hope that it might bind to the insulin receptor thereby allowing you to purify the insulin receptor. Which of the following protein separation techniques below would best accomplish this task? a. DEAE ion exchange chromatography b. Affinity chromatography c. CM ion exchange chromatography d. Gel filtration e. SDS gel electrophoresis END OF QUESTIONS RELATED TO THE ABOVE EXPERIMENTAL SETUP

51. Sirtuins and resveratrol are currently being actively studied given their potential influence on how a. They can be manipulated to differentiate human embryonic stem cells into beating cardiomyocytes b. They can be used in place of restriction enzymes c. They may influence cellular and organismic longevity d. They can influence neuronal regeneration e. They can ameliorate the progression of multiple sclerosis and other neurological diseases 52. Who below is best known for his/her ability to predict the theoretical limit of resolution of a light microscope? a. Hartwell b. Tsien c. Matthews d. Abbe e. Venter 53. The limit of resolution of a typical 100,000 volt transmission electron microscope is closest to which of the following? a. 100  m b. 10  m c. 1  m d. 100 nm e. 0.1 nm 54. Quark Pharmaceuticals and siRNA (Merck) are two companies that are using either siRNA or a combination of siRNA and antisense RNA to tackle diseases of the a. Eye b. Liver c. Pancreas d. Kidney e. Skin 55. Craig Venter a. Sails around the world on his yacht harvesting microbes looking for new genes b. Was the first to sequence the human genome c. Made the first artificial cell d. All of the above e. None of the above 56. cDNA cloning typically differs from genomic cloning given that in the former the starting material is a. mRNA b. DNA fragments c. Intact genomic DNA d. DNA with all epigenetic modifications removed e. Proteins

63. The reticular versus the neuronal theory was important to consider a. In the development of two photon microscopy b. When determining how best to prepare blood vessels for bright field microscopy c. When FACS was developed and tested d. When understanding the ability of somatic cells to limit their cell division doublings to 50. e. None of the above 64. During February there was an interesting report from Cornell that cited work done with a 3-D printer and a collagen gel that resulted in the tissue engineering construct that may be useful in the future to treat microtia. What type of construct was this? a. Engineered heart valve b. Engineered skin c. Engineered ear d. Engineered cornea e. Engineered human bladder 65. Electroporation, gold particles, liposomes and glass micropettes all can be used for a. Intracellular injection b. Cell dissociation c. Cell lysing d. Isolated human embryonic stem cells from embryos e. Plating the surface of cell culture plates to encourage cell growth 66. Cytochrome p450 function in select cell types decreases over time. This is not the case, however, with its tumor counterpart, the C3A cell used in ELADs. To what type of cell is this fact referring? a. Human skin cells b. Human pancreatic  islet cells c. Adult human liver cells d. Human skin fibroblasts e. MDCK cells 67. Complementation analysis is typically accomplished using what type of organism? a. C. elegans b. Yeast c. MDCK cells d. NHEK e. Neonatal human hepatocytes 68. When referring to the supernatant and the pellet, one is typically referencing which of the following techniques? a. Fluorescence immunocytochemistry b. Ultrastructural immunocytochemistry c. PCR d. Centrifugation e. Ion exchange chromatography

69. The experiment described in class that allowed one to use a blot to determine if the bone marrow cells in a recipient contained only those from the donor compared to those from both the donor and recipient relied on which technique below? a. cDNA microarrays b. siRNA c. Southern blots d. Northern blots e. Western blots 70. Molecular beacon probes rely on which of the following to fluoresce? a. Denaturation b. Binding of a protein c. Presence of EGTA d. Hybridization with a nucleotide e. All of the above 71. IPTG is a lactose analog used in which broad umbrella of techniques below? a. Molecular biology/transfection b. Protein purification c. Cell separation d. Stem cell biology e. Nomarski/DIC microscopy 72. If you were to inject mouse embryonic stem cells into the blastocoels of a mouse embryo you might be in the process of generating a. Monoclonal antibodies b. Polyclonal antibodies c. A knockout mouse d. A mouse that develops to the morula stage and then stops e. None of the above 73. Quantitative reverse transcriptase (RT) – PCR starts with which material? a. Proteins b. Phospholipids c. Whole genomic DNA d. Mitochondria DNA e. mRNA 74. Cluster analysis is a phrase often used when analyzing a. SDS gels using densitometry b. SELDI-TOF data c. cDNA microarray data d. Transgenic mice phenotypes e. Knock in mice phenotypes