Download artificial yeast chromosomes

ADDITIONAL LABORATORY 2 - Artificial Yeast Chromosomes
ADDITIONAL LABORATORY 2: ARTIFICIAL YEAST CHROMOSOMES
OVERVIEW
In this laboratory, you will investigate the effect of the shape and size of chromosomes on their stability. This will
be done in the context of growing various strains of yeast.
OBJECTIVES
Before beginning this lab, you should understand:
- the structure of eukaryotic chromosomes
- the process of mitosis
- the meaning of “nondisjunction”
(information on all of these can be found in chapter 12 of Campbell’s Biology, 5th Ed.)
After completing this lab, you should be able to :
- transplant yeast colonies to new plates using sterile toothpicks
- identify adenine-deficient colonies through color observation
- analyze the effects of genetic mutations on adenine synthesis
- compare the stability of artificial chromosomes versus natural chromosomes
- compare linear chromosomes with circular chromosomes in terms of stability
- quantitatively demonstrate the effect of chromosome length on stability
INTRODUCTION
Yeast are ideal experimental organisms. They are small, eukaryotic and relatively easy to grow. Furthermore,
they are not as susceptible to contamination as are bacteria (they also don’t smell as bad). Yeast can be transferred
from plate to plate using sterile toothpicks with little risk of contamination, whereas heated inoculation loops
must be used with bacteria. Yeast are particularly useful in investigating chromosomes, as in this lab, because
they are eukaryotes and thus more similar to humans than bacteria.
In this lab you will be working with several mutant strains of yeast. All of these strains have a mutation in the
gene ade2-101, which codes for adenine synthesis; this makes the yeast dependent on adenine in the external
environment and gives them a red coloration in the absence of the gene SUP11. The presence of SUP11, however,
suppresses the ade2-101 mutation, enabling the yeast to synthesize adenine and exhibit the normal white color;
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without SUP11, the yeast turn red; with one copy, they are pink, and with two or more copies they are white.
The yeast you will be working with are homozygous for ade2-101, so two copies of the SUP11 gene are necessary
to fully suppress the mutation.
The strains that you will be working with have had artificial chromosomes of varying lengths and
conformations implanted; these artificial chromosomes encode the SUP11 gene. The colonies are initially grown
in an adenine-deficient minimal medium environment, which makes the yeast dependent on the artificial
chromosome for adenine, ensuring that the artificial chromosome is not lost before the start of the experiment.
During the experiment, the yeast are grown on YPD, which has plentiful adenine; the yeast are therefore not
dependent on the SUP11 gene. Thus, when a yeast strain loses the artificial chromosome containing SUP11, it will
exhibit a red coloration due to the ade2 mutation.
Artificial chromosomes introduced into yeast cells may be lost during mitosis. Since the implanted
chromosomes are not natural, they are not as stable as normal yeast chromosomes and are more susceptible to
mitotic errors; their comparatively small size (45-140 kilobase pairs [kb], as compared with 200-5000 kb length of
natural yeast chromosomes) also contributes to these errors. There are two ways by which an artificial
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Hieter, P., Mann, C., Snyder, M., and Davis, R.W. (1985) Mitotic Stability of Yeast Chromosomes: A Colony Color Assay
That Measures Nondisjunction and Chromosome Loss. Cell 40, 381-392.
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HHS AP Biology - Laboratory Manual
ADDITIONAL LABORATORY 2 - Artificial Yeast Chromosomes
chromosome might be lost: being completely eliminated from the cell during mitosis or being unevenly
distributed to daughter cells through nondisjunction. In the former case, all cells derived from the mitotic
division where the chromosome was lost will exhibit the red color typical of the absence of the chromosome (or
pink, if one copy of SUP11 remains); in the latter case, half of the resulting colony will have the chromosome and
be white and half will lack the chromosome and be red.
The following list shows the genotypes of the yeast strains that you will be using (capital letters indicate a
normal allele, whereas lowercase letters indicate a mutant allele)
• y 262: MATa, ura3-52, lys2-801, ade2-101, trp1, his3-200
(these mutations cause a dependence on uracil, lysine, adenine, tryptophan and histidine; ade2-101
also causes a red coloration)
The following strains have the same genotype as y 262, but have also been implanted with an artificial
plasmid (plasmid lengths are given in thousands of base pairs, or kb [kilobases]):
• y 133: 45 kb circular plasmid with SUP11, TRP1 (SUP11 suppresses the red coloration)
• y 138: 90 kb circular plasmid with SUP11, TRP1 and URA3
• y 141: 90 kb circular plasmid with SUP11, TRP1, HIS3, and URA3
• y 142: 140 kb circular plasmid with SUP11, TRP1, HIS3, and URA3
• y 175: 90 kb linear plasmid with SUP11, TRP1, HIS3
• y 180: 140 kb linear plasmid with SUP11, TRP1, HIS3, and URA3
MATERIALS
~ Latex gloves
~ 4 YPD plates
~ 4 sterile toothpicks
~ yeast strains (you will be assigned 4) from:
- y 133
- y 175
- y 138
- y 180
- y 141
- y 262
- y 142
PROCEDURE: Day 1
1. Obtain 4 YPD plates. Drain any excess water into a sink, being careful to open the plates as little as possible.
2. Label each plate with your name, your group number and the number of a strain that you have been assigned
(one plate for each strain).
3. Obtain the yeast strains that you have been assigned.
4. Obtain a test tube of sterile toothpicks.
FOR EACH STRAIN THAT YOU HAVE BEEN ASSIGNED
5. Place the newly labeled YPD plate in front of you, along with the starter plate with your assigned strain.
6. Take a sterile toothpick by inverting the test tube (to get the toothpicks to the end), WITH THE CAP STILL ON.
Holding the test tube horizontally, remove the cap and carefully extract a single toothpick, being sure to touch
only the one toothpick you are taking out. Replace the top immediately after extracting the toothpick. Make sure
you touch only one end of the toothpick, and do not allow the other end to touch the table or any nonsterile surface.
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ADDITIONAL LABORATORY 2 - Artificial Yeast Chromosomes
7. Carefully dab the toothpick into a colony, being careful to open the plate as little as possible.
8. Immediately streak the yeast onto your labeled YPD plate, moving the toothpick in a squiggle pattern. Be
careful to open the plate as little as possible.
9. Discard the used toothpick into a biohazard waste container.
PROCEDURE: Days 2-8
• Observe the strains every day for the following seven days (you may have to parafilm the plates and take them
home over the weekend). Pay attention mostly to their color; they should be white, pink, red or a combination
of these colors. Record your data in the Table 1:
TABLE 1: Individual Data
1
2
Days after plating
3
4
5
COLOR(S)/PATTERN(S)
6
7
2
Days after plating
3
4
5
COLOR(S)/PATTERN(S)
6
7
STRAIN
# y_____
# y_____
# y_____
# y_____
TABLE 2: Class Data
1
STRAIN
# y 133
# y 138
# y 141
# y 142
# y 175
# y 180
# y 262
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ADDITIONAL LABORATORY 2 - Artificial Yeast Chromosomes
-4HHS AP Biology - Laboratory Manual
ADDITIONAL LABORATORY 2 - Artificial Yeast Chromosomes
ANALYSIS
Graph chromosome length vs. time to change color from white, using class averages or ideal data. Which is the
independent variable? Which is the dependent variable? Be sure to give your graph a title and label the axes.
1. What does a red coloration of a yeast colony indicate? What does a white coloration indicate?
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ADDITIONAL LABORATORY 2 - Artificial Yeast Chromosomes
2. Why were the yeast strains grown on minimal medium before the lab? Why were they grown on YPD during
the lab?
3. What role does adenine play in determining the fate of these yeast? Why?
4. What is the genetic/chromosomal difference between the red and white yeast colonies? How does it relate to
adenine?
5. What makes yeast preferable to bacteria for the purposes of studying chromosomes?
6. How might artificial chromosomes be lost from yeast cells?
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ADDITIONAL LABORATORY 2 - Artificial Yeast Chromosomes
7. Are artificial chromosomes longer or shorter than natural chromosomes? Based on your data, how does this
affect their stability?
8. Would you expect a circular or a linear chromosome to be more stable? Why?
9. Were your results consistent with your predictions (refer to your graph)? If not, how did they differ?
10. From your group’s data and class averages, what can you conclude about the effect of chromosome length on
stability (time until loss)?
-7HHS AP Biology - Laboratory Manual