STUDYING MECHANISMS OF INHERITANCE WITH DROSOPHILA MELANOGASTER

Use your lecture textbook to define the following terms before coming to lab: Sex chromosomes Autosomes Genes Alleles Homozygous Heterozygous Hemizygous Dominant Recessive Genotype Phenotype Wild-type Mutant P generation F1 generation F2 generation

INTRODUCTION Gregor Mendel is known as the father of modern genetics. In the 1860s, he published the results of his hybridization studies with Pisum sativum (garden pea) in which he proposed a mechanism for the inheritance of traits from parent to offspring. In the early 1900s, following the rediscovery of Mendels work, T.H. Morgan began a series of studies of inheritance in Drosophila melanogaster, the common fruit fly. This organism has now been used for over 100 years to study the mechanisms of inheritance in animals. WHY IS DROSOPHILA USED AS A MODEL ORGANISM FOR GENETIC STUDIES? When choosing a model organism for biological studies, there are a certain characteristics which make a model organism ideal: (1) Drosophila are small; you can house 1000s of flies in a small area; (2) they have a short life cycle (on the order of days); (3) they are easy to maintain for multiple generations; (4) they produce many, many offspring in a short period of time; and (5) they have a number of different heritable characteristics that are easily distinguishable. Week 1 Life Cycle of Drosophila How to anesthetize fruit flies: Because Drosophila can fly, they must be immobilized in order to set up your crosses and analyze progeny. If anesthetizing flies in their original culture vial, use the following procedure: 1. Obtain an anesthetizing wand. Dip the absorbent end of the wand into the Fly Nap and remove excess liquid by running the wand across the rim of the bottle.

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2. Tap the vial of flies against a hard surface so that the flies are gently knocked to the bottom of the vial. 3. Use one finger to push the vial plug slightly to the side and quickly stick the anesthetic end of the wand into the vial. (Do this quickly or the flies could escape!). 4. Lay the culture vessel on its side while the flies are anesthetized. (DO NOT leave the vial upright while the flies are unconscious or you risk drowning them in the media!) 5. Wait until the flies have stopped moving, then empty the flies out onto an index card or piece of white paper. 6. Use a dissecting scope or hand magnifying lens to examine the flies. How to determine the sex of fruit flies: You need to be able to select both male and female flies in order to have success with your crosses. Being able to determine the sex of the offspring will also allow you to see if any of the traits you are observing are sex-linked. Physical characteristics that are useful for distinguishing males and females are as follows: Body size males tend to be slightly smaller than females Tip of abdomen dark in males; lighter, striped & more pointed in females Sex combs males have sex combs on the uppermost joint of their forelegs; females do not

Figure 1: Male (left) and female (right) fruit flies [image credit: NASA]

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Fruit Fly Life Cycle: The life cycle of Drosophila can take place in plastic culture vials that contain a source of food. The life cycle has four stages: egg, larva, pupa, and adult. You should be able to see all four stages present in the cultures you will use today. The eggs are small, white found on the surface of the food. The larva hatch within 24 to 36 hours, and burrow into the food at the bottom of the vial; they look like little wriggling grains of rice. After about 5 days, they crawl out of the food onto the sides of the vial and transform into brown, oblong pupae. During the pupation stage, the larvae metamorphose into adult flies. After about 4 days, the adult flies emerge from the pupae and begin the life cycle anew. The entire cycle takes approximately 10 to 12 days to complete. You can watch an animation of the fruit fly life cycle at the following web site: http://quest.nasa.gov/projects/flies/cycleAnim.html Setting up our crosses: The purpose of this mating experiment is to determine if the inheritance pattern of the alleles that determine the eye color trait are autosomal dominant, autosomal recessive, sex-linked dominant or sex-linked recessive. This can be determined by setting up the following crosses: normal males mutant females and normal females mutant males. The offspring will be either normal or mutant depending on the pattern of inheritance. A couple of weeks ago, the following parental cross was set up: White-eyed (mutant) female red-eyed (normal) males The offspring (F1 generation) resulting from this cross are as follows: Males (): all white-eyed Females (): all red-eyed Obtain a culture vial containing flies of this F1 generation. Anesthetize the flies (as described above) and separate the males from the females using the brush provided. Check with your instructor to see if you have done this correctly. Make sure you can easily identify the eye color as this is the trait you will be observing. Transfer 3 to 5 female flies and 3 to 5 male flies to a newly prepared culture vial. Leave the vial on its side until the flies wake up. Label your vial with your group name, date and lab section.

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Week 2 Removing the F1 individuals (a.k.a., the parents of the F2 generation) Observe your culture vials for larvae and pupae. Anesthetize and remove the F1 generation from the vials; discard them in the morgue. Week 3 Examining the F2 generation Anesthetize and observe the adult flies in the F2 generation. Identify the sex and phenotype of each individual. Record your data (along with the data from the other six lab groups) in the table below. Discard the counted flies in the morgue after determining their phenotypes

Table 1: Data for Phenotypes by Sex of the F2 generation from your cross: LAB GROUP 1 2 3 4 5 6 7 8 Class Total MALES RED-EYED WHITE-EYED FEMALES RED-EYED WHITE-EYED

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Analysis of the Results: Using the data in the table above, answer the following questions: 1. Which trait is controlled by a dominant allele: red or white eyes?

2. Which trait is controlled by a recessive allele: red or white eyes?

3. Does this pattern of inheritance indicate that eye color is autosomal or sex-linked?

4. What are the genotypes of the individuals who were in the P generation? ________________ __________________

5. What are the genotypes of the F1 generation? ________________ __________________

6. Determining phenotype ratios: a. In the space below, calculate the expected ratio of phenotypes in the F2 generation.

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b. What is the actual ratio of red-eyed to white-eyed flies?

c. How does the actual ratio compare to the expected ratio?

d. If there is a difference, propose an explanation for this difference?

7. In the F2 generation, what proportion of each phenotype (red-eyed or white-eyed) was probably a. Homozygous?

b. Heterozygous?

c. Hemizygous?

8. Male to female ratios: a. What was the expected male to female ratio?

b. What is the actual male to female ratio?

c. Why were you asked to keep track of the number of males and females?

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