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					                                                                  Name_________________
                                                                  Section________________
                                                                  Spring 2007

                            Biology 212 General Genetics
                   Lab 6: The Genetic Control of Fruit Fly Eye Color

Purpose:

         To use paper chromatography to separate the pigments that determine eye color in
fruit flies. You will then use this data to determine the biochemistry and genetic control
of enzymes that catalyze various steps in this pathway.

Source of Lab:
http://www.cofc.edu/~petersj/SMFT639_Drosophila_Eye_Pigment_Lab.html

Materials:

· Chromatography paper – cut into 4x4 inch pieces.
· 600 ml beakers
· Drosophila melanogaster cultures (1 culture each of wild type, white eye, and sepia eye
flies; other cultures for testing)
· Fly Nap – for anesthetizing flies.
· glass stir rods – use the head for rubbing fruit fly eyes into the chromatography paper.
· Forceps or brushes for separating flies.
· Dissecting microscope
· UV light source
· 30 ml Chromatography solvent (1 part isopropanol to 1 part ammonium hydroxide).
Pour about 1 cm deep in the bottom of beakers in hood.

Background:

    In this lab, you will study the biochemical pathway that produces the pigments that
give fruit flies their characteristic eye color. The strains of fruit flies we will work with
are

      Wild type - red eyes
      Sepia mutant - brown eyes
      White mutant - white eyes

The biochemical pathway that produces the various eye pigments in wild type flies is
shown on the next page. Names of chemical intermediates are given; numbers refer to
enzymes that catalyze the conversion of one intermediate to another. Eye color mutants
are defective in one enzyme in the pathway that you will try to deduce from your results.




                                               1
                       precursor (colorless)

                                     1




                                     2
                                                                        7


               3                               5
                                 4




                                                    6




        Your task is to determine which pigments are present (or missing) in the mutants
and in wild type flies. You can also test for the presence of pigments in any other fly
stock (miniature, apricot, ruby, ebony, Adh, yellow). We will use paper chromatography
to separate the eye pigments.

Chromatography procedure:

1. Work in pairs.
2. Get 3 anesthetized flies from each the phenotypes (wild, white and sepia). Anesthetize
another type of flies you wish to test.
3. Get a piece of chromatography paper. Be sure to handle it on the edges only, since oils
from your hand can affect the movement of pigments on the paper. Using a pencil, draw
a line 2 cm from the longer edge, then make 4 marks along the line at about 2cm
intervals. Orient and label the paper as shown below.




                            -----+-------+-------+-------+-----
                            Wild Sepia White Test strain


                                               2
4. Use a pencil to place your initials and your partner's initials in the upper right hand
corner.
5. Using forceps to hold a wild type fly on the line at the leftmost tick, crush the head of
the fly onto the paper with a glass rod.
6. Allow the crushed heads to dry briefly (blow gently on them to speed the process).
7. Repeat Step 5 with a second wild type fly, crushing the head on the same spot as you
did the first.
8. Wash the glass rod and dry it with a Kimwipe or paper towel.
Repeat Steps 5- 8 with the two mutant flies and a strain you want to test on the
appropriate places on the paper. Be careful to crush the heads precisely on (not above) the
line at the appropriate ticks.
9. After the heads have dried for about 5 minutes, fold the paper in half between sepia
and white spots.
10. Place the chromatography paper in one of the beakers of solvent in the hood. Insert
the chromatography paper with the line of crushed heads at the bottom of the jar. Cover
the container loosely with foil (Light causes the pigments to break down).
11. Leave the paper in the beaker for 60 to 90 min. While you are waiting, you can start
to answer questions 8, 9 and 10 below.
12. Observe the pigments under UV light either immediately or the next day.

Analyzing Results: Worksheet to hand in by next week's lab (20 pts):

1. Look at the results for the wild type flies. All four of the pigments should be
distributed on the chromatogram roughly as is shown in the figure below. Using a pencil,
circle each pigment spot, and label it (Adjacent spots may blend together).

2. Using the results from the wild-type flies, compare the separated pigments for the 2
eye mutants (sepia and white) and the other mutant stock. Circle and label the pigments
for each on you chromatogram.

3. Draw a replica of your chromatograph using colored pencils in the space below



violet-blue

green-blue

yellow-green

orange

                 Wild type       Sepia        White       Mutant Strain

                                                      _____________________




                                             3
4. Summarize your results in the table below.

                                                           Mutant Strain:
               Pigment         Wild type Sepia White

               violet-blue
               green-blue
               yellow-green
               orange


               Indicate the amount of pigment using the following codes:

                                     - = no pigment
                                     + = small amount
                                   ++ = moderate amount
                                   +++ = large amount

5. Based on the results above, which of the enzymes in the pathway (indicate by
number) is probably not working properly in the sepia flies. Explain.




6. Based on the results above, which of the enzymes in the pathway is probably not
working properly for the white eye flies. (Hint: remember that mutations are very rare, so
one mutation is much more likely than many mutations!) Explain.




7. Which mutant strain did you test?                     Based on the results of your test
mutant strain, are any of the enzymes of the pigment pathway defective? Explain your
answer.




                                            4
8. The sepia and white eye phenotypes are due to recessive alleles of genes. Do these two
genes interact? The following problems will help you answer this question. Let's say that
the gene that controls the production of enzyme 4 is symbolized with a se+ = dominant, se
= recessive. The recessive allele codes for a non-functional enzyme product.

a. Can an individual that is se+se+ or se+se produce the orange pigment (drosopterin)? If
so, what will be this fly’s phenotype? (Assume that Enzyme 1 is functional).



b. What will be the phenotype of a fly that is se se? (Again, assume Enzyme 1 is
functional).



c. The gene that causes white eyed flies is X-linked recessive. Wild type is symbolized
with a Xw+ = dominant, Xw = recessive. The recessive allele codes for a non-functional
enzyme product. Can female flies that are Xw+Xw+ or Xw+Xw produce any pigments? If
so, what will be their phenotypes?



d. What is the phenotype of a fly that is XwXw?


9. a. Now let's put the two genes together. Determine the phenotypes of the following
flies:

                               genotype           phenotype
                               se+se+Xw+Xw+
                               se+seXw+Xw
                               seseXw+Xw
                               se+seXwXw

b. Now try to determine some possible genotypes of individuals with the following
phenotypes:

                                    female             male
                    Phenotype
                                    genotypes          genotypes

                    White eyes



                                             5
                    Sepia eyes



                    Wild type



c. From the tables on the previous page, explain what effect the white eye gene (w) has
on the expression of the sepia gene (se).




10. A sepia female that is heterozygous for the white eye gene is mated to a white eyed
male that is heterozygous for the sepia gene. The following results are produced:

                        Females: 26 wild type; 27 sepia; 46 white
                        Males: 23 wild type; 25 sepia; 52 white

a. Diagram the genotypes of the parents and offspring of the cross, using the gene
symbols provided above.




                                            6
b. Based on this cross, develop a hypothesis regarding the inheritance of these traits.
Briefly state your hypothesis.




c. Carry out a Chi-square test to determine if your hypothesis is correct.

                             observed-              (observed-           (obs-exp)2/
phenotypes observed expected
                             expected               expected)2           expected
wild type
sepia
white
                                                                         χ2 =

Degrees of freedom=_________________

Chi-square critical value at probability = 0.05 is ___________________

Would you accept or reject your hypothesis?_____________________




                                             7

				
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