Lab 8:  Chromatography of Drosophila melanogaster eye pigments

 

There are two classes of pigment molecules found in Drosophila melanogaster eye cells:  the ommochromes and the pteridines.  (Ziegler, 1961.)  Because the pteridine pigments are soluble in mixtures of ammonium hydroxide and n-propyl alcohol, they have been extensively studied by chromatography.  The different pigments can be separated out on paper chromatograms by their size and chemical properties.  Each individual pigment can be seen under fluorescent light, and a ratio-to-front (Rf) value can be calculated for each molecule.  (The Rf value for each pigment will vary for the individual chromatogram.)  (Hadorn, 1962.)

 

In this lab, you will be preparing a chromatogram of the eye pigments of 6 different D. melanogaster strains with different phenotypes for eye colour.  You will identify the pteridine pigments present in each of the strains, and calculate an Rf value for each pigment.  There should be six types of pteridine pigments visible in the chromatogram for a wild type fly:  drosopterins, isoxanthopterin, xanthopterin, sepiapterin, 2-amino-4-hydroxypteridine, biopterin, and isosepiapterin.  (To see the appearance and mobility of these pigments, look at the diagrams posted in the lab or at the Hadorn, 1962 reference – on reserve in the library.)  Mutant strains may be missing one or more of these pigments, or may show different amounts than the wild type strain.

 

Determining the sex (Lab 9 Figures 1, 2 and 3 – posted in lab and on website)

 

Because some of the genes involved in controlling eye colour in D. melanogaster are sex-linked, differences are seen in the amounts of pigments present in male and female flies.  Thus, each group doing this lab will have to use either all male or all female flies for your chromatogram.  There will be a total of 6 petri plates for the entire lab, each containing dead flies from one of the different strains being used.  These plates will contain both male and female flies, so the flies will have to be separated out by gender before the chromatograms can be set up.  The two sides of each double-sided bench will decide which one will use male flies, and the other side of the bench should work with the female flies.  (The one-sided bench will decide for themselves which gender to work with.)

 

Females are usually larger (7 abdominal segments vs. 5 in the males) and are more pointed in the abdomen.  In the males the posterior part of the abdomen on the dorsal side is solid black, while females have striped segments on the dorsal-posterior surface of their abdomens (Figure 1).  Males have a small brown cuticular structure (genital arch) at the end of their abdomens (Figure 2).  Also, males possess sex-combs (a black row of thick bristles) on the first pair of legs (Figure 3).  The black abdomen of the male may be difficult to distinguish if the flies are newly emerged, however, the sex-combs are always present.  Mutant body colour phenotypes, either light or dark coloured, can make the abdomen colour difference difficult to use.

 

After you are finished sorting and decapitating the flies, headless flies can be discarded in the biohazard waste buckets, and intact flies should be returned to the plates they were provided in.

 

Materials:

 

Per lab:

 

6 strains of D. melanogaster:

1.  wild type

2.  rosy

3.  sepia

4.  brown

5.  vermilion

6.  white

UV light box

 

Per group:

 

5 X 7 inch rectangle of Whatman no. 1 filter paper

small glass rod, beaker, aluminum foil, index card

~ 70 ml solvent: 1:1 mixture of 28% NH4OH and n-propyl alcohol

 

Methods:

 

1.         Label a 5 X 7 inch rectangle of Whatman no. 1 filter paper by lightly pencilling (not inking) a line ½ inch from one of the long (7 inch) sides of the paper.  Lightly mark the line with dots at ~1 inch (or 2.5 cm) intervals. Be careful not to touch the surface of the paper with your fingers, as this may affect the chromatographic properties of the paper. 

 

2.         The entire lab will be sharing 6 petri plates of flies with different coloured eyes.  Choose one of the 6 phenotypes.  Confirm that the flies are not moving, and tap a small number out onto an index card.  (If flies in the plate are moving, alert one of the TAs immediately.)  Sort out the flies by gender, until each group has 2 individual flies of the gender they have chosen.  (A dissecting microscope may aid in identifying the genders.)

 

3.         Decapitate the 2 flies on the index card (using a scalpel or dissecting pin).  Transfer both of the heads to one spot on the filter paper, and crush with the end of the glass rod.  Record which colour has been assigned to each dot.

 

4.         Wash the rod with 1:1 28% NH4OH : n-propyl alcohol after crushing the heads.

 

5.         Return the unused flies to the plate they were obtained from, and reclose it.  Discard the bodies of the decapitated flies in the biohazard waste containers.

 

6.         Repeat steps 2-5 for each of the six eye colour phenotypes.  Make sure that all flies used by each group are of the same gender.

 

7.         Prepare a developing chamber by covering one of the large beakers provided with aluminum foil.  (Pteridine pigments are light-sensitive, so the chromatogram should be developed in the dark.)  Prepare an aluminum foil lid for the chamber.

8.         Transfer the developing chamber to the fume hood indicated in the pre-lab talk.  Add 50 ml of solvent to the container, cover it, and leave it to equilibrate for 5 minutes

 

9.         Staple the short (5 inch) sides of the paper together so they do not overlap.  This should produce a tube with the crushed fly heads on the inside near the bottom.  Label the tube with a pencil near the edge.

 

10.       Place the chromatogram in the developing chamber.  (The fly heads should be at the bottom, and the solvent should not cover the fly heads.)  Make sure that the chromatogram does not touch the sides of the developing chamber.

 

11.       Close the container securely.  Develop for 90 minutes.

 

12.       After 90 minutes, remove the chromatogram from the solvent.  Mark the furthest position reached by the solvent.  Dry in the box in the fume hood for 5 minutes.

 

13.       Remove the staples from the dry chromatogram and observe it under UV light.  (Be sure to use UV goggles if box is opened during this procedure)  Mark the position of the centre of each band visible on the chromatogram for each phenotype, and note the size and colour of each band for each phenotype.

 

Report

 

There will be a short format report for this lab.  You should identify which pigments are present in each phenotype, and note differences in amounts of the pigments from the wild type.  For each pigment, calculate the Rf value for each of the pigments on your chromatogram.  This is calculated by dividing the distance from the baseline (the location of the fly heads) to the centre of each pigment spot by the distance from the baseline to the solvent front.  This value will vary between chromatograms, but should be consistent for each chromatogram, and may be useful in identifying which pigments are missing from mutant strains.

 

References:

 

Hadorn, E.  (1962).  Fractionating the fruit fly.  Scientific American, 206:  100-110.

 

Ziegler, I.  (1961).  Genetic aspects of ommochrome and pterin pigments.  Advances in Genetics, 10:  349-403.