Drosophila Eye Color
The goals of this experiment are:
To use Drosophila eye color as a model system to study biochemical pathways
To use experimental data to determine the order of steps in a biochemical pathway
To use results from genetic crosses to demonstrate the multigenic inheritance of eye color
In this lab, you will perform paper chromatography to observe the presence of eye pigments in the different eye color strains. As a class, we will design a genetic cross to study gene interactions.
Background: The Drosophila species has a compound eye made up of approximately 800 repeating units called ommatidia (singular, ommatidium). Each ommatidium contains eight photoreceptors and eleven accessory cells, including pigment cells.
Early experiments with Drosophila eye color mutants were instrumental in the field of genetics. T. H. Morgan was the first to characterize the “white eye” (w) mutation, and subsequently Beadle and Tatum extended his work to include many other eye mutant types. The significance of his work demonstrated that a change in a gene (mutation) may affect the structure, function, or regulation of a protein, in this case, an enzyme. Eye color mutants have a defect in one or more enzymes required for the biochemical pathways of pigment synthesis. As a consequence, a pigment may be missing, and/or a different pigment may accumulate because of a defect in a pigment biosynthesis pathway. The work of these eye color mutants resulted in the “one gene: one enzyme” hypothesis, which was one of the early foundations for the development of molecular biology during the second half of the 20th century. We will study a few of these eye color mutants in this experiment.
There are two classes of pigments in the eye of Drosophila, ommochromes and pteridines. Ommochromes are brown pigments, which are the products of tryptophan metabolism and pteridines are red pigments, which are the products of purine metabolism. Originally isolated in butterfly wings, pteridine pigments are bicyclic nitrogenous bases that give Drosophila eyes their color.
The rich red wild-type eye color in Drosophila is due to a mixture of several different pigments. If there is a mutation in the ommochrome (brown pigment) pathway, the semi-dull effect of the brown pigments will be missing and the eye color will be brighter red. If there is a mutation in the pteridine pathway, it will result in a duller, darker color. Pteridines are present in many invertebrates, in plants, and even in certain fishes and amphibians. These compounds are easily separated by thin layer chromatography and, when viewed under ultraviolet light, produce fluorescent spots on the chromatogram.
Chromatography of Fly Eye Pigments:
Experimental Procedure:
Eye pigments from a variety of mutant Drosophila will be separated via the technique of paper chromatography and compared with wild type eye pigments.
Chromatography
The technique used in this lab is a type of chromatography. Chromatography is a process in which compounds are separated by applying the compounds to a stationary material such as paper, and passing a moving solvent over the compounds. Depending on the relative properties of the stationary phase and the moving solvent, compounds will move fast, move slowly, or remain tightly bound at the point where they were applied. After a period of time, the process is stopped and the separated compounds are observed.
In this lab, our stationary phase will be a special paper called Whatmann paper. The moving phase or solvent passes over the paper. The solvent used in this lab is able to dissolve some substances that would not dissolve well in water. Such substances will move fast up the Whatmann paper.
Equipment
UV illuminator
Supplies
600 ml beakers(glass)
Glass rods or plastic pestles
tweezers
metric rulers
lead pencil
Whatmann Paper
Procedure
Record the phenotypes of the available flies in your notebook.
IN THE FUME HOOD, pour the solvent into a beaker to a depth of 1 cm. Cover the beaker with the provided cap or foil.
Measure 1.5 cm above the bottom of the Whatmann Paper and 1 cm from the edge, and lightly (do not gouge the paper!) using a lead pencil only, mark the spot with an x. Measure over from that x on the same line 1.0 cm and make another x. Repeat until you enough x’s for each phenotype.
Preparing the sample: It is best if you remove the heads of the flies to place on the Whatmann paper.
Obtain 3 flies of each phenotype. Be sure to keep the phenotypes separate!
Under the microscope, describe the eye color for each Drosophila line compared to wild-type Drosophila. (Is the eye color darker or lighter than wild-type?; Does it have more of an orange hue, reddish hue, brownish hue?)
Next, examine 3- 4 flies of each eye color phenotype under the microscope. Using a razor blade, cut off their heads.
Repeat for the remaining phenotypes, keeping each strain on separate sorting cards.
Using tweezers, remove the heads of the Drosophila and place on one of the X’s on the Whatmann paper
Using the glass rod, gently but firmly squash each head onto the X.
Before switching to another fly phenotype, clean the glass rod with a Kimwipe.
Carefully tape the Whatmann paper to a craft stick. Place the tape sideways, not longways, so that the tape does not overlap much of the paper.
Place the Whatmann paper into the solvent so that the bottom edge dips into the solvent and the craft stick balances on the top of the beaker. Make sure that you do NOT submerge the spots where the flies are located. Ideally the solvent should be about halfway between the bottom and the line of fly eye pigment spots. You can add or take away solvent at this stage if needed.
When the solvent moves to within 2 cm of the top of the Whatmann paper, remove the paper and wrap with a piece of aluminum foil. (The pigments are light sensitive)
Observe the Whatmann paper in bright light. Take a picture to be used in the postlab.
Observe the Whatmann paper using a UV light source. Take a picture to be used in the postlab.
After the observation and experiments, please place flies in the “morgue”.
Results and Postlab assignment:
In the space below, paste the pictures you took of the filter paper (both bright light and UV). Annotate the pictures to indicate which eye color was used for each sample on the filter paper.
Record the results in the table below for each line. The wildtype sample should have a “+” in each row. For the mutants, if you cannot see a colored pigment, use “–“. Use “+” if the pigment is present, and use “++” if there is an increase in pigment compared to wild type.
PTERIDINE
COLOR
Wild-type
White
Rosy
Sepia
Brown
Isosepiapterin
Yellow
Biopterin
Blue
2-amino-4-hydroxypteridine
Blue
Sepiapterin
Yellow
Xanthopterin
Green-blue
Isoxanthopterin
Violet-blue
Drosopterin
Orange
As mentioned in the introduction, the pteridine pigments are observed under the UV light. Using the pathway shown below, explain why each phenotype gives the chromatography results you observed.
Fly Pigment Pathway:
rosy
rosy
XDH
XDHNormal Wild Eye Type (Red Eye Fly)
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.
(image from: Genotype to Phenotype: Investigating Eye Color Mutations Using Chromatography, by Tara C. Thiemann, Truman State University http://www.public.asu.edu/~thoffman/commonfiles/lsc348/lsc348drosophilaeyepigment.pdf )
How would you determine if the sepia eye color mutation is sex-linked? Write out the crosses and use Punnett squares to show the potential progeny.
Define epistasis.
White is epistatic to sepia. Using the white and sepia flies, describe a cross that you would expect to demonstrate epistasis. Write out the crosses and use Punnett squares to show the potential progeny.
References and Resources:
Modified from http://departments.oxy.edu/tops/Flyeye/flyeyereference.htm
FlyBase: http://flybase.org/
The Interactive Fly:
Brody, T. (1999). The Interactive Fly: gene networks, development and the Internet. Trends in Genetics 15 (8): 333-4. 10431196