Teacher Guide: Analyzing the Products of DNA: Chromatography of Fruit Fly Eye Color

(adapted from Laboratory Outlines in Biology, (1994) P. Abramoff & R.G. Thomson)

Introduction

Eye pigmentation in fruit flies (Drosophila) is genetically controlled. Drosophila have genes that encode pigment-forming enzymes that make the following pigments from pigment precursor molecules.

How fruit fly eye color is made:

Some questions to get you thinking about today's lab:

How can we tell when an organism is mutant or has a mutation?

Materials (students work in groups of 4)

• Whatman filter paper #1 (others will work as well), cut in 4 inch squares
• 1:1 solution of isopropanol (2-propanol) and ammonium hydroxide (you'll need about 30 mls of the solution per group)
• glass or metal rods (1 per group)
• Drosophila, wild type (called Oregon Red and mutants (mutants available are: rosy, sepia, white, brown,cinnabar, vermilion, prune, eyeless)
• pencils (1 per group)
• ruler (1 per group)
• glass beakers (1 per group)
• aluminum foil (enough to cover beaker for each group)
• UV lamps (long wavelength)
• paper towels (1 per group)

Procedure

1. Draw a pencil line across one end of the filter paper 1 centimeter from an edge
2. Fold ends of paper in so it can stand independently with the line across the bottom.
3. The next step is to mush 1-2 flies on the pencil line, so each member of the group should mark where on the line they will mush their flies. Which flies do you want to use? Everyone should use Oregon Red (wild type) as a positive control (you can see the normal amount of all of the pigments), and then each group can choose which mutants they want to examine.
4. Once everyone has written which fly they are putting where, mush 1-2 flies on each spot. Why can we mush the whole fly and not just the eyes? Only the eye pigments are soluble in our solvent, the body pigments are not. Chromatography separates molecules by solubility and size, only the eye pigments are soluble.
5. Stand paper with pencil end down in a beaker with just enough isopropanol/ammonium hydroxide solution so that the pencil line is NOT sitting in the solution. What does the isopropanol/ ammonium hydroxide solvent do? Why don't we want the pencil line to be soaking in the solution? The solvent dissolves the pigments as it rises up the paper, allowing the pigments to rise also according to their molecular weight (small molecules travel farther than large molecules). if the flies were sitting in the solvent as opposed to above it, the pigments would just diffuse into the solvent instead of diffusing up the paper.
6. Cover the beaker with aluminum foil. Why? The pigments are UV-sensitive so they will fade if exposed to light.
7. Let the chromatograph run until the solution reaches the top, soaking the paper entirely (usually takes about 2 hours, can run overnight). What would happen if we took the paper out of solution before then? The pigments would not have had enough time to separate according to size. If you leave the paper until the pigments can't travel any more (when the solvent reaches the top), they will be spread out as much as possible. They could spread out even more if you use longer pieces of filter paper.
8. Take the paper out, letting it dry on a paper towel covered in aluminum foil (usually takes about 1 hour).
9. Examine your Drosophila eye pigments!

Examining the Drosophila Eye Pigment Data

Use a UV light to examine your filter paper chromatogram (be sure to wear protective eyewear if you are using short wavelength UV light!).

What does the pigment look like? Draw a picture of your chromatogram.

The different pigments should be in smears, some mutants will be missing certain pigments. Your students can figure out which ones are missing which.

Mark in the table which mutants have or are missing which eye pigments.

This table is drawn to mirror where the pigments will end up on the chromatogram. The orange should be at the bottom, and the yellow at the top. This will help your students figure out, for example, which of the blue pigments is missing in which mutants.

Pigment Table

Pteridine (color)	Wild Type	Mutant 1	Mutant 2	Mutant 3	Mutant 4
Isosepiapterin (yellow)
Biopterin (blue)
2-amino-4-hydroxypterin (blue)
Sepiapterin (yellow)
Xanthopterin (green-blue)
Isoxanthopterin (violet-blue)
Drosopterin (orange)

What can you conclude about your Drosophila mutants from your eye pigment chromatogram data?

Specific mutants will be missing a pigment or a group of pigments. These data mean that that mutant is missing the gene for the enzyme that makes that pigment (or group of pigments). Students should be able to refer to the pigment synthesis table and their data and determine which mutants are missing which enzymes and, thus, must have mutations in the genes coding for those enzymes. Remember that this is a biochemical pathway, so if a mutant is unable to make a pigment, the precursor for that pigment may accumulate (bigger smear in the chromatogram) and the other pigments downstream of that precursor may also accumulate. Students need to determine not only which pigments are missing, but also which pigments may be more abundant.

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BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona September 15, 2000 Designed by: Erin Dolan Nadja Anderson, Ph.D. nadja@email.arizona.edu http://biotech.biology.arizona.edu