Molecular Cell Biology Final EXAM QUESTIONS AND CORRECT ANSWERS(VERIFIED ANSWERS)|AGRADE

Molecular Cell Biology Final EXAM QUESTIONS AND CORRECT ANSWERS(VERIFIED ANSWERS)|AGRADE Separation of most blood cells is difficult, if not impossible, to achieve because they have similar properties and/or densities. What procedure is used to separate T-cells of the immune system from the many other different types of white blood cells or spleen cells? What feature of the T-cell facilitates the isolation protocol? Describe how this procedure is performed. - ANSWER- Flow cytometry is used to separate cells that have similar densities and properties. This method involves marking a cell by expressing a fluorescent protein that is specific to the desired cell type. Once tagged, the cells can be analyzed using a flow cytometer which is used to measure the light emitted as the cells pass through a laser beam. Consequently, the flow cytometer quantifies the cells within the mixture that emit the specific fluorescent protein. Using the above concept and foundation, a fluorescence-activated cell sorter (FACS) both analyzes and sorts the few fluorescent cells from the thousands of others within a mixture into a new culture dish. This procedure commonly separates T-cells from many other white blood cells or spleen cells. Only T-cells of the immune system have both CD3 and Thy1.2 surface proteins which allows the cells to be isolated using the FACS procedure. A fluorescent dye is linked to the antibodies specific to the T-cells' surface proteins and then the cell mixture is incubated with that dye. The monoclonal antibodies bind to the CD3 and Thy1.2 surface proteins making the T-cells fluorescent. The cell mixture is then mixed with a buffer in order for the cells to pass single file through a laser beam. The light emitted by each cell is measured which allows for the determination of the size and shape of each cell. Droplets, containing a single cell, are given a negative electric charge proportional to the fluorescence from the earlier measurement of the laser beam. Therefore, the nonfluorescent cells are not given a charge and the fluorescent cells have a proportional negative charge. The droplets, then, pass through an electric field where those with differing electric charges are separated and collected while those without a charge are discarded. Thus, the fluorescent T-cells are isolated from other white blood cells and spleen cells. Each organelle has a specific function in the eukaryotic cell. What determines the function of each organelle? How do integral proteins travel from the ER to the Golgi and from the Golgi to the plasma membrane? - ANSWER- The membrane and the interior space of each organelle have a a unique set of proteins and environment that enable the organelle to carry out its specific function. The interior space can hold specific enzymes that catalyze reactions within the organelle while the phospholipid membranebound proteins can control the internal ionic composition. These features separate the organelle from the surrounding cytosol and create a unique environment which consequently determines the function. Membrane and organelle proteins, in addition to all proteins secreted from the cell, are synthesized by ribosomes on the rough endoplasmic reticulum. Once synthesized, the integral proteins are transported through the rough ER's membrane via transport proteins embedded within the ER's membrane. The newly synthesized proteins accumulate in the lumen and then leave the organelle within a small membranebounded transport vesicle that was formed from the regions of the ER not coated with ribosomes. The vesicle carries the proteins to the Golgi complex. Following a series of enzyme-catalyzed chemical modifications in the Golgi complex, the integral proteins destined for the plasma membrane are transported by a second set of vesicles that have arisen form the Golgi complex. Fixatives such as formaldehyde are routinely used in certain types of electron microscopy and light microscopy. However, fixatives may introduce complications in analysis of the resulting images. What problems may result from using fixatives? - ANSWER- Fixatives cross-link most proteins with nucleic acids. In the case with formaldehyde, covalent bonds link amino groups on adjacent molecules resulting in the stabilization of protein-protein and protein-nucleic acid interactions within the cell. Thus, it renders the molecule insoluble and stable for further manipulations. Once fixed, the tissue sample is often dehydrated and embedded in a paraffin medium. After the block containing the specimen hardens, it is thinly sliced into sections. The chemical changes and dehydration caused by the fixation process may alter the shape, structure, and spatial relationship of many cellular components. These results must be accounted for during the analysis of the resulting image under a light microscope. What advantages do fluorescent dyes provide in comparison to chemical dyes that are used for staining microscopy specimens? - ANSWER- Fluorescent staining supplies a more versatile technique for localizing molecules within a cell and for visualizing specific proteins in live cells. Fluorescent compounds, or those that absorb light at one wavelength and emit light at a longer wavelength, are able to bind to specific molecules within a cell with minimal interference on the cellular shape and function. Chemical dyes often alter the internal cellular components and relationships. The variety of capabilities of fluorescent staining has over chemical staining explains the shift from the older, once common, chemical methods to fluorescent microscopy. For example, ion-sensitive fluorescent dyes can measure intercellular ion concentrations, immunofluorescence microscopy uses antibodies to localize specific components in fixed cells, and the fusing of GFP (green fluorescence protein) to a protein of interest allows its localization and dynamics to be explored in a live cell. There are many other techniques developed using fluorescent dyes as a result of their flexibility and adaptability over chemical dyes. Describe differential centrifugation and equilibrium density-gradient centrifugation. - ANSWER- Centrifugation techniques allows for the separation of proteins and nucleic acids with similar methods differentiating various organelles. Most fractions of a cell are first organized by size using differential centrifugation. This procedure begins when a filtered cell homogenate is spun at increasingly higher speeds. After each spin, the supernatant is poured out leaving the larger sediment, or organelles, collected at the bottom of the tube. The pelleted fractions collected following each spin typically contain multiple organelles of similar sizes. For instance, the nuclei are separated first following the slowest spin because they are one of the largest organelles within the cell. the next fraction amassed would contain mitochondria, chloroplasts, lysosomes, and peroxisomes. With increasingly higher speeds, the plasma membrane and microsomal fragments would be separated from the soluble portion of the cytoplasm. Once the above impure fractions have been obtained through differential centrifugation, equilibrium density-gradient centrifugation can further purify the solutions and fully separate the cellular components according to their densities. Following the resuspension of an individual fraction, the contents would be layered on top of a solution gradient of a dense non-ionic substance like sucrose or glycerol. The tube is then centrifuged at a high speed, about 40,000 rpm, for multiple hours. While the mixture is spinning, the particles and organelles migrate to a position within the solution where the density of the surrounding liquid is equivalent to the density of the particle. The individual layers, containing the purified organelles, are then recovered. How does the wavelength of the light used to illuminate a specimen affect the ability to resolve objects within the specimen? Why are chemical stains required for visualizing cells with basic light microscope? - ANSWER- The wavelength of light is proportional to the resolution of a microscope based on the equation for resolution: D= 0.61λ / N sin α . In the equation, D represents the numerical value of the resolution or the minimal distance between two distinguishable objects; consequently the smaller the value of D, the better the resolution of the microscope. The wavelength of the incident of light is represented by λ and since the D and λ are directly proportional, the resolut