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Eye color mutations chromatography drosophila

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  • 1. Genotype to Phenotype: Investigating Eye Color Mutations Using Chromatography By Tara C. Thiemann, Truman State University (B.S. Honors Biology - 2001) Background It probably was in one of your very first biology classes when you learned that DNA is a genetic map and that DNA determines phenotype. Not long after this you learned that, through the processes of transcription and translation, DNA codes for the production of proteins. Both of these concepts are conceivable alone, but perhaps their relationship has eluded you. How can the production of proteins determine phenotype? The answer is enzymes. In the 1940’s, George W. Beadle and Edward L. Tatum, while working with the mold Neurospora, determined that DNA regulates cellular chemical reactions by controlling the synthesis of enzymes. Beadle and Tatum’s one-gene- one-enzyme hypothesis suggested that the information for the production of one enzyme comes from one gene and that a mutation of this gene may render the enzyme inactive. Without an active enzyme, the biochemistry and thus the phenotype of an organism is altered. It often takes several enzymes working together in an elaborate biochemical pathway to produce a substance that alters phenotype. In Drosphila two such pathways contribute to the eye color of the flies. The ommochrome pathway produces the brown eye pigments, and the pteridine pathway produces the yellow, red, and ultra- violet pigments. This lab will focus mainly on the 7 products of the pteridine pathway, which are shown in Figure 5.1. “Despite the many investigations carried out over many years, a real understanding of the interactions leading to the production of the wild type eye color phenotype in Drosophila melanogaster remains elusive.” (Reaume, Knecht and Chovnick, 1991). In other words, the exact mechanisms of the pteridine and ommochrome pathways are not known by your Biology 107 instructor — or by anyone else! However, researchers have built a working model (Figure 6.2) that seems to yield reasonably accurate predictions for how the two pathways work. Figure 5.1 — Seven pigments produced by the pteridine pathway.
  • 2. The actual pteridine and ommochrome pathways are substantially more complex than the model suggests. For example, some individual arrows actually represent a series of several reactions catalyzed by different genes. (Refer to the background section of the enzyme lab to become more familiar with the action of enzymes.) The following is a specific example of an enzyme-catalyzed reaction in the pteridine pathway. PDA Synthase 6-pyruvoyl [sepia] Pyrimidodiazepine tetrahydropterin (PDA) Figure 5.3 – Enzyme-catalyzed reaction. The gene sepia codes for the production of the enzyme PDA synthase. Since PDA is a precursor to the drosopterins (Figure 5.2), a mutation in sepia prevents the production of these red eye pigments and increases the production of the yellow pigment sepiapterin. The gene is named sepia because of the eye color resulting from its mutation. Similarly, the genes vermilion and scarlet code for enzymes involved in the ommochrome pathway. Mutations in these genes prevent the production of the brown pigment xanthommatin, resulting in bright red eyes. Not all mutations that affect eye color affect enzymes in the pigment pathways. For eye pigments to be made, several steps must occur. First, the starting substrate must be transported into the cell. Second, the pigments must be made within the cell using the enzyme pathway. Third, the pigments must be transported to the pigment granules, which are similar to lysosomes. The protein encoded by the brown gene affects the transport of the starting substrate (GTP) into the cell. The white gene affects the transport of both GTP (starting substrate for pteridine production) and tryptophan (starting substrate for ommochrome production). These proteins belong to a larger class of transport proteins whose other members include the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and multidrug resistance proteins in cancer cells. The CFTR protein is involved in the transport of Cl- ions, and the multidrug resistance proteins transport hydrophobic drugs out of cells, making them resistant to the effects of these drugs. Figure 5.2 — Current model of the pteridine and ommochrome pathways. Genes (pink) encode transport proteins (orange) and enzymes (green rectangles). Enzymes catalyze specific reactions (green arrows) that convert chemical precursors into pigments. Pigment colors are shown in parentheses; underlining denotes pigments visible only under UV light.
  • 3. The pteridines can be separated using a paper chromatography method developed by Hadorn and his research partner Herschel Mitchell in 1951. According to this method, the fruit flies are crushed onto a piece of filter paper that is in turn placed into a solvent mixture of propyl alcohol and ammonia. The capillary action of the paper pulls the solvent upward, and as the solvent passes through the crushed flies, the pteridines are dissolved. Since the structures of the various pteridines differ, they have distinct chemical and physical properties. These distinct properties allow the pigments to be carried different distances in the paper, thus separating the pigments. In this lab you will use chromatography to analyze the number and amount of pteridines found in homozygous wild (w+), white (w), brown (bw), scarlet (sc), sepia (se), and vermilion (ve) flies. Isosepiapterin (Yellow) Beaker Biopterin (Blue) 2-Amino-4-Hydroxypteridine (Blue) Filter Paper Sepiapterin (Yellow) Xanthopterin (Green-Blue) Crushed Fly Isoxanthopterin (Violet-Blue) Solvent Drosopterins (Red-Orange) Figure 5.4 – Diagram of Hadorn and Mitchell’s chromatography method. Objectives 1. Think about the role of enzymes in the production of pteridines, and understand how this relates genotype to phenotype. 2. Identify the pteridine pigments found in Drosophila with various eye-color mutations. 3. Gain experience using paper chromatography to separate organic molecules.
  • 4. Experimental Procedures Week 1 Because the sepia gene codes for an enzyme that converts A into B, we might reasonably predict that a mutation in this gene would increase the concentration of A while decreasing the concentration of B. We might also expect this same mutation to decrease the concentration of all pigments that are downstream of B in the metabolic pathway. 1. Using (diagrams X), predict the effect of each eye color mutation (w, bw, sc, se, vm) on the concentration of each pigment. Record your predictions in Table 5.1, using the following symbols: ++ : Much more than wild-type + : More than wild-type = : The same as wild-type – : Less than wild-type –– : Much less than wild-type Table 5.1 — PREDICTED concentration of pteridine pigments in Drosophila with different eye colors. Pigment Color Wild White Brown Scarlet Sepia Vermilion Isosepiapterin Yellow = Biopterin Blue = 2-amino-4- hydroxypteridine Blue = Sepiapterin Yellow = Xanthopterin Green-blue = Isoxanthopterin Blue-violet = Drosopterins Orange = 2. Cut a rectangle of Whatman No. 1 filter paper 15 × 20 cm in size. Draw a line in pencil parallel to and 2 cm away from one of the long edges. Mark this line at 3-cm intervals, making a total of 6 marks. (See Figure 5.5, at right.) 3. Etherize 4 flies of the same eye color. With the aid of a dissecting microscope, decapitate the flies with a razor blade. Crush the 4 heads onto one of the marks on the filter paper using a glass rod. Wash the rod with n-propyl alcohol. Discard the bodies in the fly morgue at the front of the lab. 4. Repeat Step 2 for each of the five remaining eye colors. To avoid contaminating your sample, wash the glass rod with solvent between each crushing, and do not touch the filter paper with your fingers. Let the spots dry for 5-10 minutes. 5. Staple the ends of the filter paper together so that it forms a cylinder with the Drosophila heads making a ring (facing out) around the bottom. The ends of the filter paper should not overlap. Place the cylinder into a 1000- ml jar with the heads down, and carefully note the height of the Drosophila heads with respect to the jar. Then remove the filter paper from the jar. Figure 5.5 — Chromatogram Setup.
  • 5. Perform the following steps in the fume hood. 6. Dispense 50-75 ml of the solvent into the jar. The level of solvent should be about 1 cm lower than the noted position of the crushed heads. Place the lid on the jar and let the solution sit for about 5 minutes to build up vapor pressure within the jar. 7. Remove the lid and place the cylinder (heads down) into the jar. The paper must not touch the sides of the jar, and the solvent must not touch the heads. If the solvent does touch the heads, remove the filter paper, dump the contaminated solvent, add new solvent to the jar, and replace the filter paper. 8. Place the lid on the jar and wrap foil around the entire jar to prevent light from damaging the pteridines. 9. Allow the chromatography to run for approximately 6 hours. Remove the paper from the jar and let the chromatogram dry in a well-ventilated area for several minutes. The chromatogram will be kept in the dark prior to viewing in next week’s lab. Week 2 1. View the chromatogram with UV light. For each pigment listed in Table 5.2, record whether that pigment is present in your wild-type flies. 2. Compare the observed concentration of each pigment in the five eye-color mutant lines (white, brown, scarlet, sepia, and vermilion) to the concentration in the wild-type flies. Record your observations in Table 5.2, using the same symbols as in Table 5.1. 3. Compare your predictions in Table 5.1 to your observations in Table 5.2. Evaluate how accurately the pathway model shown in Figure 5.2 allows you to predict the effects of each mutation. Table 5.2 — OBSERVED concentration of pteridine pigments in Drosophila with different eye colors. 4. Explain why flies that produce high concentrations of bluish pigments do not show any trace of blue in their eye color. (Hint: Do these pigments appear blue when viewed under visible light?) 5. Explain why flies that produce almost exactly the same amount of each pteridine pigment may nonetheless have dramatically different eye colors. Pigment Color Wild White Brown Scarlet Sepia Vermilion Isosepiapterin Yellow Biopterin Blue 2-amino-4- hydroxypteridine Blue Sepiapterin Yellow Xanthopterin Green-blue Isoxanthopterin Blue-violet Drosopterins Orange
  • 6. References Dreesen, T.D., D.H. Johnson and S. Henikoff, 1988. The Brown Protein of Drosophila melanogaster Is Similar to the White Protein and to Components of Active Transport Complexes. Molecular and Cellular Biology 8:5206- 5215. Ewart, G.D., D. Cannell, G.B. Cox and A.J. Howells, 1994. Mutational Analysis of the Traffic ATPase (ABC) Transporters Involved in Uptake of Eye Pigment Precursors in Drosophila melanogaster. The Journal of Biological Chemistry 269:10370-10377. Hadorn, E., 1962. Fractionating the Fruit Fly. Scientific American 206:100-110. Higgins, C.F., 1992. ABC Transporters: From Microorganisms to Man. Annual Review of Cell Biology 8:67- 113. Phillips, J.P. and H.S. Forrest, 1980. Ommochromes and Pteridines, pp. 596-609 in The Genetics and Biology of Drosophila, Vol. 2d, edited by M. Ashburner and T.R.F. Wright. Academic Press, New York. Reaume, A.G., D.A. Knecht and A. Chovnick, 1991. The rosy Locus in Drosophila melanogaster: Xantine Dehydrogenase and Eye Pigments. Genetics 129: 1099-1109