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Lab 7 Eye Chromatography

Wild type Drosophila have dark red eyes due to a mixture of red and brown pigments synthesized from GTP and tryptophan. Mutations can disrupt pigment biosynthesis, resulting in different eye colors. The experiment separates eye pigments of wild type and mutant flies (white, sepia, scarlet) via chromatography to visualize differences. The mutants are each missing an enzyme in the pigment pathway, accounting for their distinct eye colors.

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175 views5 pages

Lab 7 Eye Chromatography

Wild type Drosophila have dark red eyes due to a mixture of red and brown pigments synthesized from GTP and tryptophan. Mutations can disrupt pigment biosynthesis, resulting in different eye colors. The experiment separates eye pigments of wild type and mutant flies (white, sepia, scarlet) via chromatography to visualize differences. The mutants are each missing an enzyme in the pigment pathway, accounting for their distinct eye colors.

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LinKanX
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Practical Genetics Lab.

The Genetics of Eye Color in Drosophila melanogaster


Chromatography to Identify Eye Mutations

Purpose:
The purpose of this exercise is to identify and investigate the inheritance of various eye
colors in mutant strains of Drosophila.

In Drosophila there are two classes of accessory pigments, which together give the fly its
dull red eye color. One class, the pteridines (from the Greek pteros, wing, because butterfly
wings owe their color to pteridines), is derived metabolically from guanosine triphosphate, GTP.
In this pathway GTP is metabolically broken down in about a dozen steps to yield ultimately
drosopterin, a group of five red pigments. Many of the eye pigments you will see are intermediates
in the pathway from GTP to drosopterin. The other class of pigments consists of a brown pigment,
ommochrome. This pigment is derived from the amino acid tryptophan in four enzymatic
reactions. Thus, the fly uses some of its GTP and tryptophan to synthesize the red and brown
pigments that it needs for visual acuity.

Drosophila mutants with various eye colors occupy a significant place in the history of
genetics. Thomas Hunt Morgan was the first to characterize the "white eye" (w) mutation. This
early genetic work demonstrated that a change in a gene (mutation) may affect the structure,
function, or regulation of a protein (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.

It is important to realize that 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. On the other hand, a mutation in the pteridine pathway will result in a duller, darker
color. The second critical point is that mutants were named based on their actual eye color, not
the corresponding biochemical defect. For example, the mutant "brown" has a brown eye color,
therefore, it is missing the pteridines, which, in turn, means that the mutation affects one of the
enzymes in the pteridine biosynthetic pathway.

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,
Practical Genetics Lab. 7
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.

Eye color in fruit flies is determined not just by a single protein, but by several different
proteins. Most fruit flies found in the wild (called “wild type”) have a darkish-red eye. But
mutations can occur in a fruit fly’s DNA, causing various flies to be born with white, sepia,
scarlet, or other colored eyes

Wild type is the name scientists use for the “normal” strain of an organism. The wild type
Drosophila strain has all of the enzymes in the protein network below, and the eye color results
from a mixture of all the pigments in the network. The white (w), sepia (se), and scarlet (st) eye
strains of Drosophila are each missing only one protein in their protein pathways for eye
pigments, and the missing enzyme is different between the three strains.

Fruit Fly Eye Pigment Chromatography:

Materials:

 Forceps  Alcohol for washing stirring rod


 Plastic T-shaped rods  400mL Beaker
 Chromatography paper (6.5 cm x 11  Pencil (DO NOT use pen!)
cm)
 Dissecting scopes (or microscopes or
hand lenses)  Chromatography solution (50%
 UV Lamps ammonia, 50% isopropyl alcohol) or
 Rulers 1:1 mixture of 28% ammonium
Practical Genetics Lab. 7

hydroxide (NH4OH) and n-


propyl alcohol
 Fruit flies (4 strains: white eyes, sepia
eyes, scarlet eyes, wild-type eyes).
Practical Genetics Lab. 7

Experimental Procedure:

Eye pigments from a variety of mutant Drosophila will be separated via the
technique of chromatography. Comparison with wild type eye pigments will allow
a visualization of the differences in the various mutants, all of the flies used
should be of the same gender. Use either male or female.

1. Obtain 2 flies of each strain .


2. Mark a pencil line about 2 cm up from bottom of paper. Try not to handle
the paper too much with your hands as oils from your fingers can affect
the chromatography.
3. On your piece of chromatography paper, label points on the down side line.
4. Use the glass or plastic T-rod to crush 2 flies one by one in different color.
smash completely but not smear the flies.
5. Clean rod using the paper towels and alcohol.
6. Repeat steps 4-5 for the remaining fly strains continuing for the other strains.
7. Measure 100mL of chromatography solution into your beaker.
IMPORTANT: The liquid should NOT touch the flies directly.
8. Wait until the chromatography solution has traveled nearly to the hole for the
pencil.
9. Remove the chromatography paper from the liquid lt to dry. Observe and
record your results for normal light and UV light conditions.
10. Use the table to identify the seven pteridine pigments found in wild-type
Drosophila. Complete Table by checking off which pigments are detected in
your chromatogram for the wild-type and mutant flies.

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

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

4
Practical Genetics Lab. 7

Sample chromatogram of wild-type Drosophila eye pigments

Conclusion:

Write a conclusion for the investigative question? What causes the variation in
eye color between different Drosophila fruit fly strains?

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