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Characterizing Drosophila Mutants

1994 Woodrow Wilson Collection


Using Chromatography: Student Handout

Background

The different eye color phenotypes among Drosophila result from alterations in biochemistry. George W. Beadle and Edward L. Tatum demonstrated in the 1940's that genes regulate biochemical processes by controlling the production of enzymes. Their work resulted in what is referred to as the "one-gene-one enzyme" hypothesis. In 1951, Ernst Hadorn and Herschel K. Mitchell at the California Institute of Technology outlined a procedure using paper chromatography to study and characterize the enzymatically controlled differences between various phenotypes of Drosophila.

Paper chromatography of the wild-type Drosophila results in the separation of seven pteridines. Pteridines are bicyclic nitrogenous bases originally isolated in the wings of butterflies, and also identified in the eye pigments of fruit flies. Flies with mutant eye colors have pteridine patterns that differ distinctly from the wild-type flies. Heterozygosity is easily distinguished since even though they possess a wild phenotype, the chromatogram will indicate an altered pteridine profile.

In this investigation, wild-type and several mutant varieties of Drosophila melanogaster will be characterized and distinguished according to pteridine pigments.

Materials

For each student group:

5-in. by 7-in. rectangle of Whatman No. 1 filter paper
small glass rod
3lb. coffee can with cover
1000mL beaker
50mL of solvent
Solvent: 1:1 mixture of 28% ammonium hydroxide (NH4OH) and n-propyl alcohol 2 flies (of the same sex and same eye color, per sample) (e.g. wild type, sepia [se], brown [bw], plum [Pm], scarlet [st], rosy [ry], cinnabar [cn], vermilion [v], eosin [we], apricot [wa], white [w]) Fly Nap (purchased from Carolina Biological)

For the class in general:

ultraviolet light source
stapler

Procedure

Fly Squash

  1. Take Whatman filter paper and lightly pencil a line 1/2 in. from one of the 7-in. edges and parallel to it. Mark this line with dots at 1-in. intervals.

  2. Using a glass rod, crush two etherized wild-type Drosophila on the first dot. Immediately wash the rod in the solvent.

  3. Repeat step #2 using two of each of the mutant flies selected for testing. Space the flies at 1-in. intervals as provided by the dots. Be sure to label each dot with a symbol indicating the type of fly used. Wash the glass rod in solvent after each squash.

  4. Let the spots air dry.

Apparatus Assembly

  1. Form the filter paper into a cylindrical shape that is 2-in. in diameter and 5-in. high. Staple the 5-in. sides so that they do not overlap.

  2. Pour 50mL of fresh solvent into a 1000mL beaker and place the beaker into a coffee can. (The coffee can is necessary since the lab must be run in the dark.)

  3. Insert the filter paper into the beaker so that only the tip of the paper touches the solvent, and so that the paper does not touch the sides of the beaker. The eye squashes should not be in the solvent.

  4. Allow the chromatogram to run for 90 minutes. Then, remove the filter paper from the cylinder and let it dry.

Observing the Chromatogram

  1. Remove the staples and unroll the chromatogram (be sure that the paper is dry!).

  2. Examine the chromatogram under UV light.

  3. Use Table A to identify the seven pteridine pigments found in wild-type Drosophila. Complete Table A by checking off which pigments are detected in your chromatogram for the wild-type and mutant flies.

TABLE A
Pigment Color Wild-Type se bw st w
Isosepiapterin yellow
Biopterin blue
2-amino-
4-hydroxypteridine
blue
Sepiapterin yellow
Xanthopterin green-blue
Isoxanthopterin violet-blue
Drosopterins orange

[n.b. the pigments listed in the table above are arranged in order of appearance on the wild type chromatogram with isosepiapterin migrating the farthest.]

Questions to answer while examining the chromatograms

  1. Compare your results with those obtained by other students who used similar mutant flies of the same sex. If necessary, suggest reasons why your results may have differed. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________

  2. Compare your results with those obtained by other students who used similar mutant flies but of the opposite sex. Were different results obtained? If so, why? _____________________________________________________________ _____________________________________________________________ _____________________________________________________________

  3. What might account for sex differences between the flies with respect to the pteridines? _____________________________________________________________ _____________________________________________________________ _____________________________________________________________

  4. Are you able to detect all seven pteridines in the wild-type flies? If not, offer some reasons why this is so. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________

  5. It has been reported that sepia-eyed flies lack drosopterin. Does your chromatogram confirm this observation? _____________________________________________________________

  6. Does the chromatogram of sepia-eyed flies show any pteridines present in amounts in excess of those found in the wild-type? If so, what are those pigments? _____________________________________________________________

  7. Reports indicate that sepia-eyed flies have excess amounts of 2-amino-4-hydroxypteridine and of biopterin. _____________________________________________________________

    Do your observations confirm these results? _____________________________________________________________

  8. What pteridines were detected in white-eyed flies and in the brown-eyed flies? _____________________________________________________________

  9. Reports indicate that brown and white eye mutants contain no pteridines at all! Do your results confirm this observation? _____________________________________________________________

Calculations and Results

The distance a compound is carried in chromatography is dependent on the nature of the compound and the type of solvent used. This distance is characteristic of chemical compounds, and is reported as the ratio-to-front (Rf) value, where

Calculate the values for the pteridine spots of the wild-type flies by marking with a pencil the solvent front at the time the chromatogram was removed from the chamber, and circle each pigment spot at the time the chromatogram was observed under UV light. Measure the appropriate distances and calculate the values. Record your results in Table B.

TABLE B
Pigment Distance from the
Baseline to Center
of the spot
Distance from the
Baseline to
Solvent Front
Isosepiapterin
Biopterin
2-amino-
4-hydroxypteridine
Sepiapterin
Xanthopterin
Isoxanthopterin
Drosopterins

Teacher Notes

All mutant types are not necessary for a successful lab. It is recommended that the wild-type be used in conjunction with at least two mutant varieties. Be sure that each student group uses flies of the same sex, since eye color in Drosophila is sex-linked. Alter the data tables as necessary.

Success in this investigation results from ensuring that the chromatogram is processed in a coffee can, in a darkened room, and for 90 minutes. For laboratory periods shorter than two hours, begin the run and designate a student from each group to remove the filter paper at the appropriate time. Hang the filter paper to dry overnight if necessary.

Wild-type Drosophila should produce a chromatogram possessing all seven pteridines. Mutants will provide chromatograms with varying pigments.


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