BIS 104 exam II key complete

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Unformatted text preview: a Page 1 Name _________________________________________ b c = X~GFP 1. The diagram shows a cell with 3 membrane ­bound organelles a, b, & c, all approximately 1 um in diameter and a protein, “X”, associated with the plasma membrane. The distance between a and b is 350 nm. The distance between b and c is 600 nm. In your lab you have a bright ­field microscope equipped with the following features: a light source with average wavelength = 500 nm; ocular lenses with 10x magnification; an oil immersion objective lens with 63x magnification and a NA = 0.60. Abbé equation: d = (0.61) (λ) / (n) (sin α) a. Can this microscope clearly distinguish a from b? Can it distinguish b from c? Show your calculations and units to justify your answers. (4) d = (0.61) (500 nm) / (0.60) = 505 nm, so, NO, a and b are too close together to resolve and YES, b and c can be resolved since d < 600 nm. b. What is the magnifying power of this microscope? Show your calculation and units. (2) total mag = (10x) x (63x) = 630x (diameters) c. Suppose that you mistakenly used water (n = 1.25) on the cover slip instead of immersion oil (n = 1.5) in setting up to view your specimen. What effect would this have on the magnification? What effect would it have on the resolution? (2) No effect on magnification. Resolution would be reduced (sub ­optimal) d. Using this microscope, what could you do to improve the visibility (contrast) of your specimen? (2) Specimen could be stained with contrast enhancing stains or vital dyes. e. In an attempt to see greater detail in your cell specimen, you replace the 10x ocular lenses with 20x oculars to increase the magnification. Now the specimen looks larger, but there has been no gain in resolution. What is this phenomenon called? (2) empty magnification Page 2 Name _______________________________________ 2. The cell on page 1 has been transfected with a DNA construct (promotor~X~GFP) coding for an integral membrane protein linked to GFP. To verify that protein X has been translated and inserted into the PM, you decide to examine the cells by conventional epifluorescence microscopy. Using a full spectrum (400 – 800 nm) white light source, describe the following features of the microscope that you would set up to view this particular labeled protein. (a) The light filtering properties of the excitation filter: (2) excitation filter will block all visible wavelengths other than the desired blue light (~410  ­ 480 nm) (b) The light filtering properties of the emission filter: (2) emission filter will block all wavelengths below 500 nm and above 580 nm, passing only green. (c) The light reflecting / transmitting properties of the dichroic mirror: (2) dichroic mirror will provide ~100% reflectance of exciting blue light and ~100% transmission of emitted green light. (d) For viewing this cell specimen, what is one advantage that scanning confocal fluorescence microscopy would have over conventional fluorescence microscopy? (2) confocal system would block out unfocussed fluor from each optical plane, providing a clearer, sharper image (better resolution of detail). 3. To test for lateral mobility of protein “X” you photobleach a small area of the fluorescent membrane for 2 seconds and then monitor the rate of recovery (FRAP) at 370C for 1 minute. Using the axes provided below: (a) label the Y ­axis and sketch in the results you would expect to see if “X” has relatively rapid lateral mobility, and (b) sketch the results you would expect if “X” were part of a lipid raft complex. (5) (a) (b) fluorescence fluorescence intensity intensity 40 40 20 60 20 60 time (sec) time (sec) (c) How might (i) changes in the fatty acid chain length and (ii) changes in the degree of fatty acid saturation in the PM phospholipids affect the mobility of protein “X”? (2) (i) mobility of the protein would be increased by sorter chain FAs (increased membrane fluidity) (ii) mobility of the protein would be increased by more unsaturated Fas (increased membrane fluidity) (the reverse is true for both). Page 3 Name _________________________________________ The diagrams below show two respiratory epithelial cells. The cell on the left (N) is from a normal individual. The cell on the right (CF) is from a cystic fibrosis patient. Information describing some of the cell components is provided below the diagrams. Questions 4 a – e (pages 4 &5) are based on these two cells and their components. CF N is the CFTR protein; an integral membrane protein with both internal and external domains and a mol wt of 210kDa; the internal domain consists of 375 amino acids. is the hypothesized mutant form of the CFTR protein. It accumulates in the cytosol and has a mol wt of 190kDa. is the an external peripheral protein with a mol wt of 25kDa same as normal is a lipid anchored (inner leaflet) protein with a mol wt of 95kDa same as normal Obviously, there would be many different kinds of proteins, both cytosolic and membrane associated, in a real cell. But, for the purpose of these questions, consider only these three. Page 4 Name ______________________________________________ 4. You hypothesize that chloride flux is defective in the CF cell because a mutation has resulted in a CFTR protein that is missing a 20kDa domain required for proper CFTR integration into the plasma membrane. As a consequence, a small, non ­functional version of CFTR accumulates in the cytosol of CF cells. To test your hypothesis, you begin by analyzing a total protein extract from both cell types by reducing SDS ­ PAGE. a. Sketch and label in each lane the Coomassie Blue (CB) banding pattern you would expect to see if your hypothesis is correct. Make sure you label the molecular weight for each band. (3) CF N ( ­) ( ­) 210 kD 190 kD 95 kD 95 kD 25 kD 25 kD (+) (+) b. Suppose that the mutant CF cells produce a defective CFTR protein that accumulates in the cytosol such that CF cells have 10 ­times the normal concentration of mutant protein compared to CFTR in normal cells. Sketch and label in each lane the banding pattern you would expect to see in this situation. Make sure you label the molecular weight for each band. (3) N CF ( ­) ( ­) 210 kD 190 kD 95 kD 95 kD 25 kD 25 kD (+) (+) c. To more thoroughly characterize the proteins in these cells, in two separate experiments you: 1) Subject both cell types to an ionic wash prior to SDS ­PAGE analysis, and 2) Subject both cell types to a brief protease treatment (no permeabilization) prior to SDS ­PAGE analysis. Sketch and label in each lane the results you would expect to see with CB staining of the gels. Make sure you label the molecular weight for each band. (5) ion. wash ion. wash protease protease ( ­) ( ­) ( ­) ( ­) 210 kD 190 kD 190 kD 95 kD 95 kD 95 kD 95 kD 44 kD 25 kD 25 kD 25 kD 25 kD (+) N (+) (+) CF (+) Page 5 Name _________________________________________ d. Describe how you would use an immunocytochemical procedure to further test your hypothesis that the CFTR protein is on the surface of normal cells, but not on the surface of the mutant CF cells. (8) EXPERIMENT: (list steps in chronological order) Obtain pure populations of both N and CF cells. Treat live cells with a fluor ­labeled ab against the external domain of the CFTR protein; allow time for binding. Wash away any unbound ab. Examine cells by fluorescence microscopy and compare N vs CF plasma membrane fluorescence intensity. RESULTS: If hypothesis is correct, PM fluor intensity would be greater in N cells than CF cells (may be non ­existent in CF cells). e. Assuming that the CFTR is the only integral membrane protein in these cells, what one additional piece of information about the CFTR protein could you obtain by making freeze ­fracture replicas of the normal cells and examining these replicas by scanning electron microscopy (SEM)? (2) integral protein distribution pattern: diffuse vs clustered; inner vs outer leaflet. Also, relative size. Page 6 Name ________________________________________ 5. S. De, et al. (Figure 4) cultured wild type α5β3 LNCaP cells and LNCaP empty vector control cells in soft agar for 3 days and then microscopically examined several random fields in the culture dishes. a. Describe the results they obtained. (4) size of WT colonies > control number of WT colonies > control b. What results were obtained when wild type α5β3 cells were cultured in soft agar supplemented with anti ­VEGF antibodies? (2) a 5 ­fold decrease in number of colonies compared to untreated WT c. Describe one negative control for the anti ­VEGF experiment. (2) treat WT cells with a non ­specific IgG at equivalent dose as experimental set up. 6. Fill in the blank with the term or concept that best fits the description. (12) a. Type of transport that allows the movement of D ­glucose into a cell, against its concentration gradient, in which a pre ­established Na+ gradient is present. Secondary active transport b. Type of transport that may generate an electro ­chemical gradient across a membrane. Primary active transport c. Type of transport kinetics that is characteristic of diffusion facilitated by channel proteins. linear d. Type of carrier protein that simultaneously transports two different solutes in the same direction. symporter e. Type of transport mechanism and the transporter that is often regulated by “voltage ­gating”. Facilitated diffusion via channel protein f. Type of transport mechanism and the transporter that the cell uses to import lysine into the cytoplasm. Facilitated diffusion via transporter (carrier; permease) protein BIS 104 Name _________________________________________________ Fall 2011 (last) (first) Exam II Student #: ____________________________________________ Exam #: ______ INSTRUCTIONS: 1. Print you name at the top of each page. 2. This exam consists of 6 questions on 6 pages (not counting cover sheet) worth a total of 68 points. Point values are given in parentheses. Make sure that you have a complete exam. 3. Write clearly; illegible answers will receive a score of zero. 4. Exams must be turned in by 9:55 a.m. 5. Points will be deducted for failure to follow any written or verbal instructions. 6. Please read the statements below: Authorization for public distribution (check only one box)  I authorize the return of my exam for pickup in a class bin or by general distribution during class.  I do not authorize the public return of my exam (if you choose this option you will pick up your exam directly from the instructor or TA). Honor Code Statement My signature below affirms that I wrote this exam in the spirit of the honor system of UCD. I neither received nor furnished any help during the exam. Signature ____________________________________________ ...
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This note was uploaded on 03/16/2012 for the course BIOLOGY BIS104 taught by Professor Bejjamin during the Fall '10 term at UC Davis.

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