Download Genetics Lab: Monohybrid Crosses in Yeast

Genetics Lab: Monohybrid Crosses in Yeast
AP Biology
Background
Yeast is ideal candidate for laboratory work because it has simple nutritional needs, its
growth is temperature dependent, and the entire life cycle can be completed in a couple
days.
In yeast, gametes contain eight chromosomes and diploid cells contain sixteen
chromosomes. Saccharomyces cerevisiae, Baker’s yeast is an ideal organism for studying a
complete life cycle because it can be maintained in either the haploid or diploid stages of
development, thus making the concept of genetic inheritance patterns less abstract. In
sexual organisms, the life cycle is composed of a series of developmental events that
alternate between the haploid (n) phase and the diploid (2n) phase, otherwise known as
“alternation of generations” (see figure).
The transition from the haploid to the diploid phase results from mating between opposite
sex gametes. Yeast gametes are conventionally referred to as one of two mating types: a or
alpha. A yeast pheromone released by an a cell will stimulate an undifferentiated alpha cell
to transform into a specialized gamete, and vice versa. This transformation is marked by a
change in the shape of the a and alpha cells: they each elongate and become “pear-shaped”.
These distinctive cells are known as “shmoos” (see figure).
In the process known as sexual conjugation, two “shmoos”, one a and one alpha, will
eventually fuse together at their small ends to form a characteristic “peanut” shape with a
central constriction (see figure). Within the “peanut”, the two haploid (n) nuclei fuse into a
diploid (2n) nucleus. This process forms a true zygote.
The cell division cycle begins with a single, “peanut-shaped” unbudded zygote. The zygote
reproduces by budding at the constriction. The bud grows to nearly the size of the parent
cell, forming a “clover leaf” shape. Meanwhile, within the nucleus of the parent cell, the
chromosomes replicate by mitosis, and then the nucleus eventually divides equally
between the parent and the newly budded cell, leaving each with an equal number of
chromosomes. The two cells eventually separate into two unbudded zygotes. The cycle
then begins again for both of the diploid cells, resulting in an exponential increase in the
number of diploid cells. At this rate, a single cel can grow into a barely visible colony in one
day.
The “alternation of generations” from the diploid phase (2n) to the haploid (n) phase
results from sporulation. Sporulation is a process by which the diploid nucleus goes
a gametes
Genetics Lab: Monohybrid Crosses in Yeast
AP Biology
through meiosis, producing four haploid nuclei, which are then incorporated into four
stress resistant ascospore. The spores remain together, looking much like oranges,
encapsulated in a transparent sac-like ascus. All asci should contain four spores, two of
mating-type a and two of mating-type alpha. These vegetative spores will reproduce
asexually by budding until triggered by an opposite mating-type to differentiate into a
game, which would initiate a new life cycle.
Four haploid strains of S. cervisiae will be used in the following experiment to demonstrate
basic genetic principles. The parental, or P, generation will be assumed, in accordance with
Mendel’s traditional experiment. The four haploid strains represent the gametes of the
traditional Mendelian F1 generation. A monohybrid cross will be performed among these
gametes to produce the F2 generation.
This monohybrid cross will follow two forms of one trait: red vs. white color. Mendel used a
simple, short-hand method, using capital and lower case letters, to identify the alleles in his
monohybrid crosses with peas. In a similar manner, the allele for the dominant, white color
of yeast will be represented by R. The recessive red color is then represented by r. These
haploid genotypes, R and r, can be determined by testing the yeast on a particular kind of
growth medium. The color trait in yeast is visible when it is grown on a nutritionally
complete medium (YED). The following table lists the four possible gametes and their
phenotypes on YED.
Genotype
Phenotype on YED
R(a), R(alpha)
White
r(a), r(alpha)
red
Based on the law of segregation, a Punnett square is extremely important in determining
the outcome of crosses in Mendelian genetics because it clearly displays the possible
combinations in chart form.
The simplest Punnett square to construct is one for a monohybrid cross. A good example of
this is a cross between mating-type a yeast with a red color and mating-type alpha yeast
with a white color. Determining the genotype of the yeast being crossed is vital to the
accuracy of the results of a Punnett square. If it is known that the trait for red color is a
recessive mutation, the yeast with red color must be homozygous recessive for the trait,
and therefore have a genotype rr. If you assume that the alpha yeast is heterozygous, it will
have a genotype of Rr. According to the Law of Segregation, only one of the alleles for that
trait can be passed on to the gamete from each parental yeast. Therefore, the alpha yeast
could pass either the R or r allele on to its offspring, while the a yeast can pass on only r,
thus making the only possible allelic combinations in the offspring Rr and rr. This is
diagrammed in a Punnett square below.
alpha gametes
R
r
r
Rr
rr
r
Rr
rr
The two possible genotypes, Rr and rr, will exist in a 1:1 ratio, and the phenotypic ratio will
also be 1:1 with as many offspring with red color as with white color.
Genetics Lab: Monohybrid Crosses in Yeast
AP Biology
In the following experiment, you will perform a traditional Mendelian monohybrid cross, in
which the true-breeding parental generation (P) consists of a homozygous dominant trait
crossed with a homozygous recessive trait. A cross among the P generation gametes
produces the resulting first filial, or F1, generation, which consists of 100% dominant
phenotypes and 100% heterozygous genotypes. This monohybrid cross can be
demonstrated by the Punnett square above. The diploid parents of the following
monohybrid cross are mating-type a, with homozygous genotype RR, and mating-type
alpha, with homozygous genotype rr. The F1 generation is 100% heterozygous, with
genotype Rr.
Objective
You will culture and mate a set of haploid S. cerevisiae strains to produce the diploid strains
that would be produced in the second filial generation, or F2, of the traditional Mendelian
experiment. This will demonstrate the basic concepts and principles of genetic patterns
and inheritance. You will also predict results of crosses according to the Law of Segregation
and diagram monohybrid crosses that will reinforce the idea of dominant and recessive
traits.
Materials
Subculture of yeast strain a1
Subculture of yeast strain a2
Subculture of yeast strain alpha1
Subculture of yeast strain alpha2
1 YED plate
25 Toothpicks (approximately) in clean covered container
Mating grid – tape this to bottom of YED plate
Procedure
Watch and dry hands thoroughly or put on sterile latex gloves before working with
toothpicks. Wipe down work area with alcohol to ensure a clean, sterile work environment.
Dispose of all used lab supplies according to teacher’s instructions.
Part A: Answer pre-lab questions
1. Explain, using genetic symbols, the F1 cross that you are performing.
2. Create a Punnett square to predict the combinations of alleles that will be seen in
the F2 generation. List the genotypic frequencies and the phenotypic frequencies.
3. Explain why the genotypic frequencies might be different from the phenotypic
frequencies.
Part B: Subculture F2 Haploid Strains
1. Obtain your YED agar plate with mating grid and a container of toothpicks.
2. Lift the lid from the alpha1 culture plate.
3. Using the flat end of a clean toothpick. Gently touch the haploid alpha1 colony and
gather a small sample. Replace the lid.
Important: To ensure sterile conditions, handle each toothpick by one end, and be
careful not to touch the opposite end to anything except the yeast or sterile agar.
4. Lift the lid of your YED agar plate and, using a circular motion, carefully spot your
alpha1 sample directly within the square labeled “alpha 1/R”. Be very gentle when
spotting. Do not puncture or tear up the agar with your toothpick.
Genetics Lab: Monohybrid Crosses in Yeast
AP Biology
5. Replace the lid and discard your toothpick.
6. Using a new toothpick for each strain, repeat steps 2-4 for yeast alpha2, a1, and a2.
Use your mating grid as a guideline.
7. Incubate your plate, inverted, at 30˚C for 24 hours. If an incubator is not available,
be sure to double the incubation time to 48 hours at room temperature.
Always invert plates when incubating or storing to reduce the possible contamination
of agar and cultures from condensation formed on the lid of the plate.
Part C: Prepare Diploid Mating Mixtures
1. Obtain your YED plate from the previous procedure.
2. On your observation sheet, record the colors that have developed in your Punnett
square.
3. Lift the lid of your YED plate.
4. Using a new toothpick, gather a small sample of the haploid strain alpha1 from the
square labeled “alpha1/R” on our YED plate.
5. Make a small spot in the blank square directly below the “alpha 1/R” square. Discard
the toothpick.
6. With another new toothpick, gather a sample of haploid strain a1 from the square
labeled “a1/R” on your YED plate.
7. Make a small spot of a1 next to the alpha1 spot you just made. Discard the toothpick.
8. Using a new toothpick, thoroughly mix the two spots together, using a gentle,
circular motion, being sure not to tear the agar. Make sure to keep the mating
mixture contained in the square.
Important: Make sure to mix the strains well. This will ensure optimal conditions for
mating among a and alpha strains.
9. Replace the lid of your plate and discard the toothpick.
10. Using the same techniques as with alpha1 and a1, transfer and mix samples of
alpha1 and a2, alpha 2 and a1, and alpha2 and a2 in the intersecting squares on your
grid. Remember to use a new toothpick for each transfer and mixture.
11. Invert and incubate your plate at 30˚C for 24 hours.
Part D: Compare Predictions with Observations
1. Obtain your YED from the previous procedure.
2. Record your results a Punnett square in your lab book. In each square, note whether
you see red or white.
3. Answer the following questions.
a. How did your results compare with your predictions? Use phenotypic ratios
to compare.
b. If the results from your experiment differed from your predictions, what are
some possible reasons why this error may have occurred.
c. Each organism has two factors for each of its traits. These factors are genes
and are located on chromosomes. Genes exist in alternative forms. What are
these different gene forms called? Identify the different forms in this
experiment.
d. Describe Mendel’s Law of Segregation in terms of the F1 and F2 generations
in this yeast experiment.