Download Analysis of heredity: fruit fly crosses

BC2004
Lab Exercise 7
Spring Semester 2005
Analysis of heredity: fruit fly crosses
Genetics, the science of heredity, traces its beginnings to the work of Gregor Mendel. Mendel
was unaware of DNA and of chromosomes, both of which were discovered many years after his
death. He based his inferences on the patterns of inheritance of traits that were evident in the
physical appearance of the organism – today called “phenotypic” traits.
Mendel proposed that each physical trait of an organism was the result of the action of a separate
factor (now called a gene), and that genes could occur in different forms (now alleles) that
caused a trait (e.g., flower color) to differ (e.g., purple or white) from one individual to another.
Mendel crossed and examined large numbers of plants, and his quantitative studies of garden pea
populations, generation after generation, led him to the inference that each individual possessed
two copies of the gene for a given trait, but passed only one member of the pair to each of its
offspring. Each offspring inherited one copy of the gene from each parent and so, like each of its
parents, possessed two copies.
From his hundreds of pea plants, Mendel also inferred two principles regarding the pattern of
inheritance of traits. These principles have become known as Mendel’s Laws – the Law of
Segregation (which refers to the pattern of inheritance of alleles) and the Law of Independent
Assortment (which refers to the pattern of inheritance of chromosomes, and so of the sets of
genes on different chromosomes, today called “unlinked” genes).
The Law of Segregation holds that when gametes are formed, only one copy of each gene is
present in the gamete; the two copies of a gene possessed by the parent segregate into different
gametes. If the two copies of the gene are different alleles, the alleles segregate.
The Law of Independent Assortment relates to the sorting out of genes that an individual
inherited as sets from its own two parents. As an individual forms gametes, a set of genes (i.e., a
chromosome) that it inherited from its mother may or may not end up in a gamete with any other
maternal chromosome; some of the chromosomes it inherited from its maternal parent end up in
gametes with chromosomes it inherited from its paternal parent. The assortment of
chromosomes in each gamete is a random mixture of chromosomes from the individual’s mother
and father because the maternal chromosomes assort independently of each other, as do the
paternal chromosomes.
We now know that there are exceptions to Mendel's laws, especially the law of independent
assortment. Many traits are influenced by the action of many different genes, and one gene can
influence many different traits. We also know that not every gene is present in the adult as two
copies. The most obvious exception to this rule is seen in sex chromosomes.
Sex chromosomes are not present in all organisms, but when they are, they are often of different
sizes, with one so much larger than the other that it carries many more genes than the smaller
one. In some animals, including mammals (and fruit flies), the female individual possesses two
BC2004, Spring 2005, Lab exercise 7-1
of the larger sex chromosome (called X) and the male possesses one X and one of the smaller sex
chromosome (called Y). In other animals (e.g., birds), the male possesses two of the larger
(called Z) and the female possesses one Z and one smaller sex chromosome (called W).
In many of the species with X and Y chromosomes, the Y chromosome is much smaller than the
X and carries fewer genes. Females inherit two copies of genes located on the X chromosome
(X-linked genes), but males are hemizygous (carry only a single copy). Consequently, if a male
inherits a recessive allele on his X chromosome and does not have an allele for that gene on his
Y chromosome, he will express the recessive phenotype, whereas a female can inherit a second
allele and will display the recessive phenotype only if she is homozygous for the recessive trait.
For this reason, human and fruit fly males display a higher frequency of recessive traits than do
females.
Although most of the genes carried on sex chromosomes govern traits not related to the sex of
the individual, and genes on other chromosomes influence the development of sexual
characteristics, at least one of the sex chromosomes will carry information crucial to determining
the sex of the individual. All the other chromosomes are called autosomes. Alleles of genes
carried on autosomes segregate and assort in patterns not related to the sex of the offspring.
In this exercise, you will follow the inheritance of several traits in Drosophila melanogaster, the
fruit fly. Fruit flies are useful in genetics because they grow quickly (a new generation emerges
in about 2 weeks), produce many offspring, and are easy to maintain. In addition, there is now a
great deal known about Drosophila biology, and the sequence of the entire genome was
completed in the year 2000.
Lab Exercise 7 will be divided into two sections. In the first section, you will learn to distinguish
males from females and will familiarize yourself with the traits to be studied. You will then set
up crosses by placing males and females of different phenotypes together in vials and allowing
them to mate. The vials of flies will be incubated at 25oC. One week after you have prepared
the mating vials, you will remove and discard the parents. The vials, which should now contain
only the fertilized eggs, will be re-incubated for two more weeks, during which time the
offspring of the cross will emerge from the eggs and develop into adults whose traits you will
inspect and record in order to analyze patterns of inheritance.
You are going to perform two separate experimental crosses. In the first, you will study the
inheritance of genes that influence different traits: eye color and wing development. In the
second, you will perform reciprocal crosses (wild-type female x mutant male, and wild-type
male x mutant female) to determine whether a particular trait is sex-linked or autosomal.
BC2004, Spring 2005, Lab exercise 7-2
Handling Drosophila
Fruit flies fly. They are also quite small. In order to control them while you study and
manipulate them, you will have to anesthetize them. For this purpose, you will place the flies on
a plate that delivers a low level of CO2 through diffusers, and you will observe the flies under a
dissecting microscope.
NOTE: for any procedure that the flies must survive, they must NOT remain on the
anesthetizer for longer than 10 minutes.
Have ready: vial(s) of stock flies
anesthetizer plate
dissecting microscope
small brush
marking pen
(for crosses: three fresh food vials)
To remove flies from a vial:(*)
Shake the flies away from the cotton plug into the bottom of the vial by gently tapping the
bottom of the upright vial on the lab bench; “gently” is important, to prevent the flies from
getting stuck in the food at the bottom of the vial. While tapping lightly, quickly remove the
vial’s cotton plug, invert the vial on the anesthetizing plate, and dump the flies onto the plate.
Replace the cotton plug in the vial.
To manipulate anesthetized flies:
As soon as the flies are anesthetized, observe them under the dissecting microscope. Use the
brush to gently (again) manipulate, sort and select flies. Be careful that you do not damage the
flies.
To return or transfer flies to a vial:
When you are finished studying or manipulating the flies, remove the cotton plug from the
(labeled) vial that is to receive the flies, place the vial opening close to the edge of the CO2 plate,
and gently sweep the flies into the vial, using the brush. Immediately replace the cotton plug.
If the flies are to be incubated, check them after 5 to 10 min. to be certain that they are awake
and active, then place the vial in the green tray on the instructor’s bench. If your flies have been
over-anesthetized, they will be dead. If this happens, consult with your instructor.
If a vial of flies is to be discarded, place the vial in the tray labeled “discard.” The flies will be
humanely uthanized.
To discard parents after one week (see page 7-6):
Remove the adult flies from each mating vial, as directed above (*), and sweep them into a
container labeled “fly morgue.” Return the embryonic flies, still in the mating vials, to the green
tray on the instructor’s bench. Why do you need to remove the parents after one week? Why not
after one day or after two weeks?
BC2004, Spring 2005, Lab exercise 7-3
Procedures (work in pairs)
A. Getting acquainted with fruit flies
Before setting up mating populations, you need to learn to distinguish male flies from females,
and wild-type traits from variations that are due to genetic mutations in genes that affect those
traits, so-called “mutant traits.” The life cycle and the distinguishing features of males and
females are illustrated at the end of the exercise. The traits that will be used in these experiments
are shown in the Table of Traits below.
TABLE OF TRAITS
Trait
Wild type
Appearance (allele)
eye color
Mutant
Allele
Appearance
red (P+)
Plum (P)
dark purple eyes
red (se+)
sepia (se)
medium-brown eyes
red (w+)
white (w)
white eyes
wing contour
flat (C+)
Curly (C)
wings are curled upward
wing angle
straight back (D+)
Dichaete (D)
wings extend outward at a
slight angle from the body
body color
gray (e+)
ebony (e)
black body
gray (y+)
yellow (y)
yellow body
Note that in the symbolism used in Drosophila genetics, the wild-type allele of a gene is
represented by the symbol for the mutation followed by a “+” sign.
Also note that the symbol for a mutant allele that is dominant to the wild-type allele is written in
upper case (e.g., Plum (P) is dominant to red wild type (P+), and the symbol for a recessive allele
is written in lower case (e.g., sepia (se) is recessive to red wild type (se+).
For this exercise, you and your partner will be provided at your station with six vials of flies for
practice:
i.
wild type (red eyes, gray body, and flat, straight wings)
ii. Curly wings
iii. Dichaete wings
iv. Plum eyes
v.
mixture of phenotypes
vi. one recessive mutant: yellow body, ebony body, sepia eyes, or white eyes
BC2004, Spring 2005, Lab exercise 7-4
Working with the flies from only one vial at a time:
(return each set of flies to its appropriate vial before opening another vial of flies)
1. Anesthetize and examine the wild-type flies. Separate them by sex, and have an
instructor check to be sure that you have correctly distinguished males from females
(this is very important when setting up crosses!).
2. Anesthetize and examine flies with each mutant phenotype. Work swiftly (to protect
the flies), but study each type long enough to be sure that you can recognize each
phenotype.
3. Finally, anesthetize and examine the mixed population. Separate the flies into groups
according to phenotype, and have an instructor check to be sure that you have done
this correctly.
B. Experimental crosses
From the front bench, obtain one set of empty mating vials (3 vials/set), two vials of virgin
female flies, and three bottles of populations containing both males and females. Check
that you have the following vials and bottles:
1. one set of three empty mating vials (3 vials per set)
2. VIAL of virgin female wild-type flies; these females are homozygous for all genes to be
studied.
3. VIAL of virgin female flies displaying the recessive trait assigned to you.
4. BOTTLE of male and female wild-type flies; the males are homozygous for all genes to
be studied.
5. BOTTLE of male and female Plum/Curly/Dichaete flies; the males are heterozygous for
all three genes.
6. BOTTLE of male and female flies displaying the recessive trait assigned to you.
BC2004, Spring 2005, Lab exercise 7-5
To perform each cross, first label each of the mating vials to identify each cross; add your initials
and lab section (day, time, and room number) on each vial.
Experimental cross 1
wild-type females x Plum Curly Dichaete males
(initials, lab section)
Place two virgin wild-type females in your labeled vial. Add two Plum/Curly/Dichaete males to
the vial (you will need to sort out these males; make sure they are male!).
Experimental cross 2 (reciprocal crosses)
a. Wild-type females x Mutant males (specify which mutant)
(initials, lab section)
Place two virgin wild-type females in your labeled vial. Add two males displaying the recessive
phenotype (you will need to sort out these males; make sure they are male!)..
b. Mutant females (specify which mutant) x Wild-type males
(initials, lab section)
Place two wild-type males in your labeled vial. Add two virgin females displaying the recessive
phenotype (you will need to sort out these males; make sure they are male!)..
When you have prepared all three crosses, secure your vials together with a rubber band, and
place your set of three vials in the green tray on the front lab bench.
BC2004, Spring 2005, Lab exercise 7-6
One week later (week of 3/28-4/1/05):
During this week, your flies will mate with each other, and the females will lay eggs in the vials.
In order to prevent offspring and parents from interbreeding, you will need to remove
the parents before the offspring become sexually mature.
Retrieve your three mating vials. Remove the parents (there should be four adults in each vial), and
discard them as directed. Return the vials to the green tray on the instructor’s bench.
After two more weeks (week of 4/11 to 4/15/05):
During these two weeks, the offspring will mature.
Retrieve your three mating vials. Working with one vial at a time, anesthetize the flies and sort them
by sex and by phenotype. Record the numbers in the data tables.
When all flies have been scored, return all vials to the green tray on the instructor’s bench.
BC2004, Spring 2005, Lab exercise 7-7