Lab 7
Genetics of Organisms
Introduction:
Drosophila melanogaster,
the fruit fly, is an excellent organism for genetics studies because it has
simple food requirements, occupies little space, is hardy, completes its life
cycle in about 12 days at room temperature, produces large numbers of
offspring, can be immobilized readily for examination and sorting, and has many
types of heredity variations that can be observed with low power magnification. Drosophila has a small number of chromosomes
( four pairs). These chromosomes are easily located in
the large salivary gland cells.
The Life Cycle of Drosophila
- The eggs. The eggs are small, oval shaped, and
have two filaments at one end. They are usually
laid on the surface of the culture medium and, with practice, can be seen
with the naked eye. The eggs hatch into larvae
after about a day.
- The larval stage. The worm like larvae eats most
continuously, and its black mouth parts can be seen moving back and forth
even when the larvae is less distinct. Larvae tunnel
through the culture medium when eating; thus channels are a good
indication of a successful growth culture. The
larvae molt twice as it increases in size. In the
last of the three larval stages, the cells of the salivary glands contain
giant chromosomes, which may be seen readily under low-power magnification
after proper staining.
- The pupal
stage.
When a mature
larvae in a laboratory culture is about to become a pupa, it usually
climbs up the side of the container or on to a paper strip provided in the
culture bottle. The last larval covering becomes
harder and darker, forming a pupal case.
- The adult stage. When metamorphosis is complete, the
adult flies emerge from the pupal case. They are fragile and light in color and their wings
are not full expanded. These flies darken in a
few hours and take on the appearance of an adult fly. They
live a month or more and then die. A female does
not mate for 10-12 hours after emerging from the pupa. Once
she has mated, she stores a considerable quantity of sperm in receptacles
and fertilized her eggs as she lays them. To
ensure a controlled mating, it is necessary to use females that have not
been mated (virgins).
Figure
7.1 The Life Cycle of Drosophila melanogaster
Design of the
Exercise
This genetics experiment
will be carried on for several weeks. Drosophila with
well-defined mutant traits will be assigned to you by your teacher. You are responsible for making observations and keeping
records concerning what happens as mutant traits are passed from generation to
the next.
You will be assigned to study a
certain mode of inheritance using particular genetic crosses of flies having
one or two mutations. The modes of inheritance most
commonly used are:
- Monohybrid. In these experiments, the mode of
inheritance is determined when a single contrasting pair of traits is
involved.
- Dihybrid. In these experiments, the mode of inheritance
is determined when two pairs of contrasting traits are considered at the
same time.
- Sex-linked. In these experiments, the mode of
inheritance is determined when the mutant characteristic is associated
with the X chromosome.
Procedure:
1. Obtain a vial of wild-
type flies. Practice immobilizing and sexing these
flies. Examine these flies and note the
characteristics of their eyes, wings, bristles, and antennae.
2. To make handling easier,
immobilize the flies with fly-nap, or by twirling the vial in ice packs for
several minutes. Place the immobilized flies on a
piece of filter paper inside a petri dish. Place this under a dissecting microscope to view the
flies.
3. Distinguish male flies from
female flies by looking for the following characteristics:
a). Males are usually smaller than females.
b). Males have dark, blunt abdomens, and
females have lighter, pointed abdomens.
c). Only males have sex combs. which are groups of black bristles on the upper most joint
of the forelegs.
Figure
7.2: Female and Male Drosophila
Female
Male
Male
with vestigial wings
4. Obtain a vial containing
pairs of experimental flies. Record the cross number
of the vial. This number will serve as a record as to
which cross you obtained. These flies are the parental
generation(P1) and have already mated. The
female should have already laid eggs on the surface of the culture medium. The eggs represent the first filial, F1
generation and will be emerging from their pupal
cases in about a week.
5. First week (today): Immobilize and remove the adult flies. Observe them carefully under the dissecting microscope. Separate the males from the females and look for the
mutation(s). Note whether the mutation(s) is/are
associated with the males or females. Identify the
mutation(s) and give them a made up name and symbol. Record
the phenotype and symbol in Table 7.1. The findings
should be confirmed by your teacher.
6. Place the parents in the
morgue(jar containing alcohol). Label the vial
containing the eggs or larvae with the symbols for the mating.
Also label the vial with your name and date. Place
the vial in a warm location.
7. Second week: Begin by observing the F1
flies. Immobilize and examine all the flies. Record their sex and characteristics. Consider
the conclusion s that can be drawn from these data. Place
five or six pairs of F1 flies in a fresh culture bottle and the rest
of the flies in the morgue. For this cross the females
need not be virgins. Label the vial with the symbols , name, and date.
8. Third week: Remove the F1 flies from the
vials and place them into the morgue. The F2
generation are the eggs and /or larvae in the vial. Place
the vial in a warm place.
9. Fourth week: Begin removing the F2 flies. Record their sex and the presence or absence of
mutation(s). The more F2 flies collected,
the more reliable the data will be. You may have to
collect flies over a three-or four day period. Try to
collect at least 200 flies.
10. To analyze your data, you will
need to learn how to use the Chi-Square Test. Go to
the Statistical Analysis Section to review the technique.
Table
7.1: F1 Generation Data
Date
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Phenotype and
Symbol |
Females |
Males |
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Table
7.1: F2Generation Data
Date
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Phenotype and
Symbol |
Females |
Males |
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Analysis of
Results:
1. Describe and name
the observed mutation(s).
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
2. Write a hypothesis
which describes the mode of inheritance of the trait(s) you studied. This is your null hypothesis ( as
described in the statistical analysis section).
____________________________________________________________________
____________________________________________________________________
3. Refer to a textbook
and review Punnett squares. In the space below,
construct two Punnett squares to predict the expected results of both the
parental and F1 crosses from your null hypothesis.
|
Parental
Cross |
F1
Cross |
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f |
r |
4. Refer to the Punnett squares
above. Record the expected ratios for the genotypes
and phenotypes of the F1 and F2 in the experiment below.
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Expected
Genotypic Ratio |
Expected
Phenotypic Ratio |
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F1 |
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F2 |
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5. Do the actual results
deviate from what was expected? If so explain how.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
6. From the results
describe your cross: is it
Sex-linked or
autosomal?_________________________
A dominant mutation
or a recessive mutation? ________________________
Monohybrid or
Dihybrid? ________________________
7.
Are the deviations for the phenotypic ratio of the F2 generation within the
limits expected by chance? To answer this question,
statistically analyze the data using the Chi-square analysis.
Calculate the Chi-square statistic for the F2 generation in the chart
below.
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Observed
Phenotypes (o) |
Expected (e) |
(o-e) |
(o-e)2 |
(o-e)2 e |
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X2 = |
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a). Calculate the Chi-square
value for these data.
1. How many degrees
of freedom are there? ___________________
2. Chi-square (X2)
= __________________
3. Referring to the
critical values chart, what is the probability value for these data? _____________
b). According to
the probability value, can you accept or reject your null hypothesis? Explain why.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
Topics
for Discussion:
1. Why was it necessary for the females of the parental generation to be
virgin?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
2. Why was it not
necessary to isolate virgin females for the F1 cross?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
3. Why were the adult flies removed from the vials at weeks 2 and 4?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
