Soakin’ Up the Rays with Schizosaccharomyces pombe
Laboratory Objectives
After completing these labs, you should be able to:
· Explain how UV radiation affects DNA
· Describe how DNA mutations can lead to cancer
· Define the phases of the cell cycle and identify the function of checkpoints
· Create and identify repair mutants in yeast
· Differentiate repair mutants, checkpoint mutants and wild type yeast using a fluorescence microscope
Introduction
Normal cell division is an essential process for all living organisms that is responsible for reproduction, growth and development. For example, the single cell of a fertilized human egg divides again and again to create billions of cells as it develops into a zygote into an infant into an adult. The process of cell division is regulated by the cell cycle, which functions to ensure that each newly created cell receives an identical copy of DNA (hereditary material) and the necessary components to carry out its cellular function.
Figure 1: The cell cycle.
The cell cycle is a sequence of four phases: G1, S, G2, and M (Figure 1). G1, S, and G2 are collectively called interphase and during this time the cell grows and duplicates its DNA in preparation for cell division during mitosis or the M phase. Vital to this process is the accurate replication and distribution of DNA (hereditary material) to each daughter cell. Human DNA contains over three billion base pairs, and errors can and do occur during replication. To correct for this, the cell cycle contains a control system of checkpoints that enable the cell to check on its growth and DNA replication. These checkpoints verify that the cell is ready to progress to the next phase of the cycle. If a cell is not ready, a delay in the cell cycle is initiated by the checkpoint system so the cell has additional time to correct the deficiency (Table 1). This is important so that new cells do not contain extra, missing, or incorrect portions of DNA.
Table 1: Cell Cycle Checkpoints
Cell Cycle Phase / CheckpointG1/S / Has the cell grown large enough?
Has DNA been damaged?
G2/M / Has the cell grown large enough?
Has all DNA been duplicated?
Are all chromosomes intact?
M / Are all chromosomes attached to the spindle?
Deficiencies in a cell’s checkpoint control system can result in abnormal cell division and become cancerous. Disruptions in normal cell cycle function can arise from damage to a cell’s DNA that does not get repaired. For example, ultraviolet (UV) light is a form of radiation that can damage DNA and ultimately lead to abnormal changes in the way cells function, grow and divide. Exposure to UV light from the sun is the leading cause of skin cancer and is associated with 90% of non-melanoma and 65% of melanoma skin cancers.
To gain a better understanding of how UV light can damage DNA and cause abnormal cell division, you will perform a series of experiments that will consist of exposing yeast cells to UV light in order to create “mutant” cells that are deficient in their ability to repair DNA and respond to checkpoints in the cell cycle.
Why do we study yeast to learn about cancer?
Yeast is a model organism used in many areas of scientific research, including the cell cycle. This eukaryotic organism is unicellular, divides rapidly, and contains a small genome with numerous genes that are similar to those found in a human. Schizosaccharomyces pombe is a species of fission yeast that grows by elongation at its ends and divides at the midpoint, creating two daughter cells of equal size. S. pombe is usually haploid during its life cycle, which means that it contains just one set of chromosomes. This makes it an ideal model to observe the effects of genetic mutations, as a mutant trait is readily apparent without another gene copy to mask its effects.
Part 1: Mutagenesis of Yeast with Ultraviolet Light
In this lab you will:
· Determine the yeast cell concentration in liquid culture using a hemocytometer and light microscope
· Calculate the dilution factor necessary to dilute the culture to 50,000 cells/ml
· Dispense and spread the cells on plates using proper sterile technique
· Mutagenize the cells with UV (ultraviolet) light
· Discuss DNA damage, cell cycle checkpoint system, yeast as a model organism
Materials
S. pombe yeast, rad22-gfp, in liquid culture
light microscope + video screen 6 YES media plates
hemocytometer micropipettes + tips
clicker counter Bunsen burner + sparker
Kimwipes cell spreader + glass dish
YES liquid media Sharpie marker
microcentrifuge tubes 70% ethanol
microtube holder disposable gloves
UV Crosslinker beaker for tip disposal
Note: If you are unfamiliar with micropipettes, practice pipetting with water before beginning the experiment.
Safety Information
Wear disposable gloves during the experiment.
Background Information
The yeast you are using for these experiments contain a DNA repair protein, called Rad22, which is tagged with green fluorescent protein (gfp). Rad22 is produced by yeast cells when strands of DNA are damaged and need to be repaired. The genetic code for gfp has been added to the rad22 gene. Each Rad22 protein will contain gfp, which is visible with a fluorescent microscope. You will use the gfp tag to locate and analyze the presence of Rad22 during this lab project.
Yeast grow in a liquid media called YES (yeast extract + supplements), which contains nutrients that support the growth of healthy cells. Agar is added to YES to solidify the media in petri plates. Healthy cells growing in YES will double every 2.5 hours.
A hemocytometer is a special microscope slide used to determine yeast cell concentration. A counting chamber containing a grid of perpendicular lines has been etched in the slide. Because the area and depth of the grid are known, we can use the number of cells counted on the grid to calculate the cell concentration of the yeast culture.
Procedure
Part A. Determining yeast cell concentration (# cells / ml): Work in a group of 4
1. Create a small volume of a 1:10 dilution of cells in liquid media. For example, place 900ul of liquid media into a microcentrifuge tube. Swirl the flask of yeast culture to suspend the cells and add 100ul of yeast cells to the tube. (1 tube per group of 4)
2. Set the hemocytometer flat on the table and place the coverslip so the metallic loading notch (shaped like a V) is accessible with the tip of a micropipette, see Figure 2.
3. Load the hemocytometer by dispensing 10ul of the 1:10 yeast dilution onto the loading notch. The area under the coverslip should fill completely by capillary action. If air bubbles are present under the coverslip, rinse the hemocytometer and coverslip with distilled water, dry with a Kimwipe, and reload with a new sample.
4. Place the hemocytometer on the microscope and using the 10X objective, adjust the focus until the counting grid comes into view. Take care not to crush the objective against the slide, as the hemocytometer is much thicker than a standard microscope slide.
5. Count the number of cells in each of the four outer corners of the grid, as shown in Figure 3. If two or more cells are touching each other, count them as 1 cell. Note: If more than 50-60 cells are counted in the first corner, increase the dilution to 1:20 or 1:50, clean and reload the hemocytometer and begin again.
6. Calculate the average number of cells per corner.
7. Calculate the number of yeast cells/ml in the initial liquid culture by using the formula: (average # of cells per corner) x (dilution factor) x (10,000) = # cells/ml
Figure 2: Hemocytometer (www.valdosta.edu)
Figure 3: Hemocytometer grid/counting chamber (www.free-ed.net)
Count the number of cells present in the four outer corners of the grid, circled and labeled A, B, C, and D. Each quadrant is 1mm x 1mm ; depth is 0.1mm.
Part B. Plating cells: Each student has their own 6 plates
1. To allow the yeast room to grow on the plates, the initial culture must be diluted. Calculate the volume of initial yeast culture and volume of liquid media required to produce 1 ml of final yeast culture that contains 50,000 cells. A useful formula is CIVI = CFVF (initial concentration)(initial volume) = (final concentration)(final volume).
2. Combine the volumes of initial yeast culture and liquid media calculated above in a clean microcentrifuge tube. This is the final yeast culture that is ready to be plated. Tip: Yeast are heavy and will sink in liquid media. Always swirl or mix the culture before pipetting yeast.
3. Spray the work table with 70% ethanol and wipe dry.
4. Set up the work space with a Bunsen burner, glass petri dish of 70% ethanol, cell spreader, micropipette + tips and 6 media plates. Label the bottom of each plate with your initials and the date.
5. Plate 5000 cells to each media plate.
a. Dip the cell spreader in ethanol and pass through the flame of the Bunsen burner. Important: wait 30 seconds after the flame burns out before touching the cell spreader to the yeast. A hot cell spreader will kill the yeast.
b. Invert the tube to mix the yeast. Dispense 100ul of final yeast culture onto a media plate.
c. Use the cell spreader to distribute the culture quickly and evenly across the media. Tip: keep lids on the plates whenever possible to prevent contamination.
d. Important: allow yeast culture to dry on the media before proceeding with UV radiation.
Part C. Damaging DNA with UV Radiation
1. Important: the surface of the media should be dry before beginning this procedure.
2. Place 5 media plates in the UV Crosslinker machine, remove the lids and close the door.
3. Push the “time” button, followed by 8 seconds and “start.” When time is up, replace the lids.
4. The sixth plate is not exposed to UV radiation. Label it “no UV.”
5. Stack plates upside down and store in a 30C incubator for 3-4 days.
6. Plates will then be stored in a refrigerator until the next lab class.
Post-Lab Assignment
1. What is the name and purpose of the plate that is not exposed to UV radiation?
2. Why does the class need to mutagenize ½ million yeast in order to create a DNA repair of checkpoint mutant?
3. Complete Hemocytometer and Gene Homology assignments.
Part 2: Replication of Yeast Colonies
In this lab you will:
· Replica plate the mutagenized yeast colonies from the previous class
· Discuss the use of HU+ plates (that contain hydroxyurea) and HU- plates (that do not contain hydroxyurea) as a tool to identify yeast colonies that are DNA repair and checkpoint mutants.
· Review the cell cycle, DNA repair, and the checkpoint system
Materials
6 YES plates from last class 70% ethanol
5 YES + pB media plates (HU-) disposable gloves
5 YES + pB + HU media plates (HU+) Sharpie marker
5 squares sterile velvet pail of soapy water
replica block & collar
Safety Information
Caution: hydroxyurea and phloxine B are hazardous chemicals.
Wear disposable gloves during the experiment.
Dispose of master plates in the biohazard waste container.
Background Information
To identify yeast colonies that contain DNA repair or checkpoint system mutants, the plates from the last lab must be replicated onto media plates that contain hydroxyurea (HU). HU is a toxin that damages DNA and interferes with DNA replication. The checkpoint control system of wild type (“normal”) yeast cells responds to DNA damage by delaying cell division so the DNA repair system can repair damage caused by HU. Once the damage is repaired, the cells will progress through the cell cycle. Yeast with a defective DNA repair or checkpoint system cannot divide normally and will die on media containing HU.
Replica plating is a screening tool to identify colonies with a selectable trait. The replica process transfers a genetically identical copy of cells to a plate that contains a different growth media to identify colonies that cannot grow in the new media. In this experiment, the secondary plates will contain HU so that we can identify colonies that cannot grow on HU+ media and thus have a deficiency in their DNA repair or checkpoint systems.
Phloxine B (pB) is a pink indicator dye added to the media. Healthy yeast cells take up phloxine B and then expel it as they grow and divide. Yeast that die after they have been transferred to HU+ media do not survive long enough to eliminate phloxine B and appear dark pink.
Procedure
1. Examine and compare the UV and control plates. Note similarities and differences.
2. Label the new media plates:
a. Label the bottoms with your initials and the date.
b. Organize and mark plates in sets with 1 HU- (no HU) and 1 HU+ (with HU) plate per set.
c. Make an orientation mark on the bottom outside edge of each plate. This will be used to orient the plates correctly during the replica process.
3. Make an orientation mark on the bottoms of the media plates from the first lab. These plates are called the master plates.
4. Spray the table, replica block, and collar with 70% ethanol and wipe dry.
5. Prepare the replica block by placing a velvet square over the top of the block with the fuzzy velvet nap facing up. Place the collar over the block so the velvet is stretched smooth and held firmly in place and the black mark on the collar is in the 12o’clock position.
6. Remove the lid from a master plate and turn the media plate upside-down. Align the orientation mark with the 12 o’clock position on the block and set the media on the velvet. Press gently and evenly over the bottom of the plate to leave a yeast imprint on the velvet, then remove the plate.
7. Transfer the yeast to a HU- plate. Remove the lid and turn the new media plate upside-down, making sure the orientation mark is aligned with the 12 o’clock position. Gently press media to the velvet to transfer the yeast.