Experiment I, Part III - Tripeptide and Protein Modeling 2

RESTRICTION NUCLEASE ANALYSIS OF VIRAL DNA The analysis of large macromolecules, whether proteins or nucleic acids, is considerably facilitated by techniques for subdividing the molecules into sizes which are more tractable for analysis. To be useful, the smaller fragments of the molecules must be specific, i.e., they must be produced in the same way from every molecule of their large precursors, and their relationship to the larger molecules from which they are produced must be known. As we have seen, some proteases provide a basis for such techniques for protein analysis. For nucleic acids, the corresponding agents are the restriction nucleases. These enzymes recognize and cleave DNA at specific, short sequences. Because they are sequence specific, digestion of large DNA molecules gives rise to fragments with defined sizes. Restriction nucleases have revolutionized molecular biology, not only because they permit defined subdivision of large DNA molecules, but also because the sites at which cleavage occurs are used as “mileposts” - location markers in a polymer which is otherwise structurally featureless. The locations of functional positions (genes, operators, recombination sites, etc.) can then be defined with respect to such mileposts. A first step in the characterization of a large DNA molecule is thus the development of a “restriction map” of the DNA, a linear diagram of the DNA marked with the locations of sites cleaved by various restriction enzymes. An example of such a map is shown below for the DNA from the bacteriophage . Generation of the data which give rise to such a map is straightforward: restriction enzymes are commercially available, and the restriction reaction itself and size analysis of its products are technically trivial. Analysis of these products, on the other hand, is a logical challenge. The DNA fragments produced in these reactions are analyzed by size fractionation on agarose gels. Agarose is a thermotropic medium: dilute solutions (ca 1%) are liquids at temperatures above about 40°C, but become relatively solid at lower temperatures (just like Jello, although the interactions are somewhat different). Once separated, the DNA fragments can be visualized (at sub-microgram levels) by soaking the gels in ethidium bromide. This planar, polycyclic molecule binds to DNA by intercalation between the DNA base pairs; when it is intercalated, the dye fluoresces a bright orange upon excitation with UV light. In this experiment, we will attempt to generate a restriction map for the DNA molecule which is the entire genome of bacteriophage . We will use the restriction sites for the restriction enzyme EcoR1 (from Escherichia coli) as our reference point for analysis of the maps for BamH1 (from Bacillus amyloliquifaciens H), Hind III (from Hemophilus influenza D), and Xho I (from Xanthomonas holicola). 27 PROCEDURES: Restriction of DNA The reaction mixtures for restriction of the DNA should each be made up on ice in the following order: 32.5l 5 l 2.5l 5 l 5 l H2O 10x concentrated incubation (not electrophoresis) buffer DNA (0.2 g/l) enzyme #1 enzyme #2, if there are two, or H2O Be careful to make each addition to the bottom, rather than on the walls, of the tube. Change tips to avoid cross-contaminating reagents! Mix the contents by gently “flicking” the tube with your finger. If droplets are left on the wall, a brief centrifugation will drive them to the bottom of the tube. When all of the samples are ready, and securely labelled, transfer them to the 37° bath and incubate for 60 minutes. At the end of this time, stop the reactions by the addition of 5 l of “stop” solution (150 mM EDTA; 50% glycerol; 0.1% bromphenol blue). Finally, unless directed otherwise, heat the stopped reactions for 5 minutes at 65°C and quick-cool (plunge the tubes into ice water). Pouring the Gel Mix 180 ml of H2O, 20 mls of 10x concentrated electrophoresis buffer (500 mM Tris-HCl, pH 7.8; 60 mM sodium acetate; 10 mM EDTA) and 1.6 g of agarose. Dissolve the agarose by bringing the solution just to the boiling point, swirling, reheating, swirling, etc., until the solution is completely clarified. Remove from heat and allow the suspension to cool. While the agarose solution is prepared, the electrophoresis tray should be prepared as well. To this end, tape across the open ends of the “gel trays”, folding the corners neatly so that the seal is watertight. Seal the seams by dripping a bead of agarose from your hot agarose solution along the tray/tape seams, using a Pasteur pipette. Place the sealed tray on a flat surface, position the well former so that it is about 1 inch from one end of the tray and raised 1 mm above the glass surface. Pour the moderately cool agarose into the tray and allow it to set. Prepare 1 liter of electrophoresis buffer (900 ml of water plus 100 ml of the 10x concentrated electrophoresis buffer, well mixed) and pour some of this buffer into the gel apparatus. Remove the support from the wellformer, leaving the wellformer itself sticking into the gel. Remove the tape from the ends of the gel tray, put the gel tray into the electrophoresis tank, and add more buffer until the gel is submerged to a depth 28 of 2-3 mm. Finally, gently tease out the wellformer. Electrophoresis When all of the digested DNA samples are ready for electrophoresis, prepare some blank samples by mixing 90 l H2O and 10 l of “stop” solution. Practice loading 30 l of this solution into the wells at the edges of the gel. then load your various DNA samples into the remaining wells, avoiding the outermost wells. Note carefully the order in which your samples are loaded, and choose your wells to give an asymmetric pattern of loaded wells (so that you can recognize which sample is which, even if the gel is upside down). Cover the apparatus, connect the leads so that the DNA will migrate through the gel towards the anode (positive) side, and turn on the power, setting the voltage to 30 V. The gels will be run overnight, and the TA will remove them in the morning and stain them by soaking in a solution of 0.5 g/ml ethidium bromide. Once stained, the gels will be photographed. Data Analysis On the day following the laboratory, photos of the gels will be posted on the course website. Using the Eco R1 fragments as molecular weight markers, assign sizes to all of the various visible fragments. Using the double digestion data, and any partial digestion fragments which you can identify, each group should draw a map of the genome, indicating the location of as many sites as can be tentatively assigned. The logic used in making the site assignments should be laid out in your notebook as clearly (and briefly) as you can manage. The lab grade for this exercise will be assigned primarily on the basis of the clarity and brevity of this last item, not on the accuracy of the map. Incubation Buffer The ideal incubation buffer is different for different restriction enzymes, principally in the amount of salt present. Our digestions will all be performed in: “1” x incubation buffer: 50 mM Tris-HCl pH 8.0 100 mM NaCl 10 mM MgCl 2 29 Digestions Groups should prepare samples of DNA digested as follows: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 30 Eco R1 BamH1 or Hind III, depending on assignment Xho I Eco R1, no 65° treatment BamH1 (or Hind III), no 65° treatment Xho I, no 65° treatment Eco R1 (once more, for luck) Eco R1 and BamH1 (or Hind III) Eco R1 and Xho I Xho I and BamH1 (or Hind III) Undigested DNA (no enzyme) A bit of Log scale graph paper for sketching a Log (Mol Wt) vs distance plot: 31 TRANSPORT LAB Introduction The hydrophobic character of the plasma membrane endows it with its essential physiological property, impermeability to the common water soluble, lipid-insoluble molecules. While this is an asset in the retention of the cellular machinery and metabolic intermediates, it offers an obstacle to entry of essential nutrients. In order to overcome this difficulty, cells have evolved special transport mechanisms involving specific protein molecules which recognize and translocate small molecules across the hydrophobic barrier of the plasma membrane. The simplest of these mechanisms involves a type of protein which facilitates the movement of a specific molecule across the otherwise impermeable membrane and allows the equilibration of concentrations on the two sides. No metabolic energy is required for this process; net movement is driven by the energy stored in the concentration gradient across the membrane. this process has been called facilitated diffusion or carrier mediated transport. The best studied example is the glucose transport system of the human erythrocyte. Glucose entry into most animal cells (with the exception of the renal tubule and intestine) occurs via this same mechanism. Examples of facilitated diffusion of carbohydrates have been described in yeast and E. coli. glucose uptake by facilitated diffusion is seen in yeast during anaerobic alcoholic fermentation. Efficient flow of glucose into this cell is maintained by the rapid hydrolysis of polysaccharides by externally located enzymes leading to a high local concentration of glucose near the plasma membrane. The great advantage of facilitated diffusion to the cell's economy is that no metabolic energy is required for the operation of the carrier. The primary disadvantage of such a mechanism over active transport is the inability of facilitated diffusion to scavenge nutrients at low concentrations from the environment. Thus, survival by cells possessing this primitive transport mechanism depends on relatively high concentrations of nutrient in the external medium. One may speculate that many organisms have found such facilitated diffusion systems either entirely unsuitable or inefficient when required to extract substances in trace amounts in the external environment. Under these conditions a definite selective advantage was conferred upon those organisms which evolved transport systems capable of efficiently scavenging low concentrations of nutrients in the medium. E. coli, for example, found low concentrations of lactose in their natural environment of the lower gut of mammals. Scavenging this metabolizable sugar presumably gave these organisms a significant selective advantage over their neighbors. While an expenditure of metabolic energy is required for active transport, it must have been outweighed by the advantages accrued from the ability to utilize this additional nutrient. 33 Bacterial Coupled Transport In 1963 Mitchell(1) proposed that microorganisms couple the movement of protons down their electrochemical gradient with the uphill membrane transport of various substrates (substrate-proton symport). According to the chemiosmotic hypothesis, cells extrude protons either as a result of oxidative electron transport (aerobically) or as a result of the activity of a membrane-bound ATPase (anaerobically). Such proton extrusion results in both a membrane potential (inside negative) and a pH gradient (inside alkaline). The energy stored in this electrochemical gradient of hydrogen ions is converted into active transport of the substrate by coupling of the flows of the two substances. According to Mitchell's nomenclature, the electrochemical force or protonmotive force (p) is equal to the sum of the electrical potential across the membrane () and the potential due to the pH gradient (expressed in electrical units). At 25° and with pH equal to the outside pH minus the inside pH, the relationship would take the following form: pmv = mv -59mvpH A still-current model for the transport processes in bacteria like E. coli, S. lactis, and S. faecalis is diagrammed below.(2-4) 34 In bacteria that can function aerobically, oxidation is thought to drive a pump which pushes H+ to the outside of the cell. Thus, metabolic energy is used to establish a pH-gradient and most likely a membrane potential () as well. Glucose, for example, can ultimately provide the energy to run the proton pump during oxidation. In turn, the protonmotive “force” (p) generated as a result of oxidation can be used for the phosphorylation of ADP, or can be used to drive the transport of metabolites into the cell against concentration gradients. (Notice the similarity to the oxidative phosphorylation process in the mitochondrion.) Under anaerobic conditions glycolytic degradation of glucose forms ATP which can drive the membrane-bound ATPase in the reverse direction to produce a pH gradient and a  (ATP hydrolysis makes the inside of the cells alkaline and negative in potential relative to the medium). This membrane energy or protonmotive energy can be used for the active transport of metabolites into the cell. References **Abstracted from the following papers: T. H. Wilson, E. R. Kashet and M. Kusch. (1972). The Molecular Basis of Biological Transport. Miami Winter Symp. 3 (eds. J. F. Woesner and F. Huijing), Academic Press. E. R. Kashket and T. H. Wilson. (1973). Proc. Natl. Acad. Sci. 70:2866. 1. Mitchell, P. (1963). Molecule, Group and Electron Translocation Through Natural Membranes. Biochem. Soc. Symp. 22:142-168. 2. Maloney, P. C., E. R. Kashket and T. H. Hastings. (1974). A Protonmotive Force Drives ATP Synthesis in Bacteria. Proc. Natl. Acad. Sci. 71:3896. 3. Kaback, H. R. (1986). Active Transport in E. coli: Passage to Permease. Ann. Rev. Biophys. Biophys. Chem 15:279-319. 4. Harold, F. M. (1986). The Vital Force: A Study of Bioenergetics. W. H. Freeman. Esp. Ch. 5. 35