· Web viewThe 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

Soakin’ Up the Rays with Schizosaccharomyces pombe

Laboratory ObjectivesAfter 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

1 Soakin’ Up the Rays

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 CheckpointsCell 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

MaterialsS. pombe yeast, rad22-gfp, in liquid culturelight microscope + video screen 6 YES media plateshemocytometer micropipettes + tipsclicker counter Bunsen burner + sparkerKimwipes cell spreader + glass dishYES liquid media Sharpie markermicrocentrifuge tubes 70% ethanolmicrotube holder disposable glovesUV 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 InformationThe 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 41. 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 Assignment1. 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

Materials6 YES plates from last class 70% ethanol5 YES + pB media plates (HU-) disposable gloves5 YES + pB + HU media plates (HU+) Sharpie marker5 squares sterile velvet pail of soapy waterreplica 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 InformationTo 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.

Procedure1. 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.7 Soakin’ Up the Rays

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.

8. Duplicate the replica process using a HU+ plate, making sure the orientation mark is aligned with the 12 o’clock position.

9. Remove the collar and place the velvet in a pail of soapy water.10.Repeat the replica procedure for each of the master plates.11.Stack the pairs of new replica plates upside-down and store in a 30C incubator for 2-3 days.

Plates will then be refrigerated until the next class.12.Dispose of the old master plates in the biohazard waste container.13.Each set of HU- and HU+ plates will be examined during the next class to locate a mutant

yeast colony that can grow on HU- media but cannot grow on HU+ media.

Post-Lab Assignment

1. What differences did you observe between the UV and control plates? Offer a possible explanation(s) for your observations.

2. Explain what is similar about all the yeast in one colony. How may a colony be different from neighboring colonies?

3. Explain at the molecular level why some yeast colonies will be able to grow on HU- media but will die on HU+ media.

4.


Part 3: Identification of DNA Repair and Checkpoint Mutants

In this lab you will: Compare yeast colony growth on HU+ and HU- media Identify possible DNA repair and checkpoint mutants Transfer potential mutants to new growth media

Materials10 replica plates from last class 70% ethanol1 YES media plate disposable glovesPetri dish reference grid Sharpie markersterile toothpicks magnifying glass

Safety InformationCaution: hydroxyurea and phloxine B are hazardous chemicals.Wear disposable gloves during the experiment.Dispose of replica plates and toothpicks in the biohazard waste container.

Background InformationReplica plating the colonies exposed to UV radiation onto media containing DNA-damaging

agent hydroxyurea allows us to identify yeast that have a mutation is their DNA repair or checkpoint systems. Possible mutant yeast can be located by comparing colony growth on HU+ media to growth on HU- media according to the characteristics in Table 2.

Table 2: Characteristics of living and dead yeast colonies

Characteristic Living Colony Dead Colony

Color light pink or white dark pink

Shape rounded, bubble-like flat

Sheen shiny, wet dull, dry

Procedure1. Spray the table with 70% ethanol and wipe dry.2. Write your name and the date on the YES plate. Make a reference mark on the bottom, outer

edge of the plate.3. Place the YES plate on a Petri dish reference grid, aligning your mark to arrow on the grid.4. Organize your replica plates in HU-/HU+ sets with orientation marks aligned in the 12 o’clock

position. Compare one set at a time.


5. Look for corresponding yeast colonies that have died on the HU+ plate but are still alive on the HU- plate. Use the characteristics listed in Table 1 to identify colonies that contain possible mutants.

6. Using a sterile toothpick, gently scoop the mutant colony (but not the media) from the HU- plate and transfer it one of the squares on the YES plate. Tip: check to make sure the YES plate is properly aligned on the reference grid so potential mutant colonies don’t get mixed together.

7. Repeat the process with the remaining sets of plates. If you are unsure about a colony, go ahead and transfer it.

8. Incubate plate upside down at 30C for 2-3days. Plates will then be refrigerated until the next class.

Post-Lab Assignment

1. Explain why you transferred the yeast colonies from the HU- plate and not the HU+ plate.2. UV radiation mutates DNA in random locations throughout the genome. Why don’t we see

other types on mutants on the plates?


Part 4: Verification of DNA Repair and Checkpoint Mutants

In this lab you will: Replica plate the potential mutants from Part 3 Verify that the selected yeast colonies are DNA repair or checkpoint mutants

Materials1 plate of potential mutants from last class 70% ethanol1 YES + pB media plate (HU-) disposable gloves1 YES + pB + HU media plate (HU+) Sharpie marker1 square sterile velvet pail of soapy waterreplica block & collar

Safety InformationCaution: hydroxyurea and phloxine B are hazardous chemicals.Wear disposable gloves during the experiment.Dispose of the master plate in the biohazard waste container

Procedure1. Label the new media plates with your initials, the date and HU+ or HU-. 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.

2. Make an orientation mark on the bottom of the master plate from Part 3. 3. Spray the table, replica block, and collar with 70% ethanol and wipe dry.4. Replica plate the master plate of potential mutants using the same procedure from Part 2.

a. 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 12 o’clock position.

b. Remove the lid from the 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.

c. Transfer the yeast to the HU- plate. Remove the lid and turn the 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.

d. Duplicate the replica process using the HU+ plate, making sure the orientation mark is aligned with the 12 o’clock position.

e. Remove the collar and place the velvet in a pail of soapy water.5. Incubate the replica plates upside-down in a 30C incubator for 2-3 days. Plates will then be

stored in the refrigerator until the next class.6. Dispose of the master plate in the biohazard waste container.7. Your instructor will transfer mutant yeast to liquid culture for Part 5.


Part 5: Analysis of DNA Repair and Checkpoint Mutants

In this lab you will: Compare and contrast wild type and mutant yeast Observe DNA and repair protein Rad22 using a fluorescence microscope Classify mutants as DNA repair or checkpoint control

Materialsreplica plates from Part 4 disposable glovesmutant yeast from Part 4 P20 micropipette + tipsyeast slides: wild type & mutants microscope slides + cover slipslight microscope microscope oil + lens paperfluorescence microscope Sharpie marker

ruler

Safety Information Caution: hydroxyurea and phloxine B are hazardous chemicals.Wear disposable gloves.Dispose of replica plates in the biohazard waste container.

Background InformationWild type and mutant yeast cells respond differently when exposed to DNA damaging agents.

Hydroxurea (HU) is a toxin that damages DNA and stalls DNA replication. Wild type yeast have a functioning checkpoint and repair systems and will delay cell division so DNA can be repaired before the cell progresses through the cell cycle. The yeast mutants created with UV radiation are defective in their checkpoint system or their DNA repair system and will respond to HU differently than wild type yeast. You will analyze wild type and mutant yeast to determine the characteristics of checkpoint and repair mutants and identify which type of mutant(s) your class created.

A fluorescence microscope allows us to visualize DNA and DNA repair protein Rad22 in the yeast cells. Rad22 has been tagged with green fluorescent protein (gfp) and will glow bright green. We can use this green protein to locate Rad22 in the yeast and determine how DNA damage affects its location and concentration. DNA is stained with DAPI and will appear bright blue under the fluorescent microscope. DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to A-T rich regions in DNA.


ProcedurePart A: Light microscope comparison of wild type and mutant yeast morphology

1. Obtain prepared slide sets for wild type yeast and two mutants. There is an untreated slide (HU-) and a slide treated with hydroxyurea (HU+) in each set.

2. Observe the untreated and treated wild type yeast with the light microscope at 40x magnification. Examine them for length, shape, and overall condition; noting differences in single cells, cells that appear ready to divide, or have recently divided.

Why should we look at wild type yeast before observing the yeast mutants?

What differences do you observe when comparing treated (HU+) to the untreated (HU-) wild type yeast?

3. Examine both the wild type and mutant slide sets (HU- and HU+), observing at least 25 cells on each slide. Using a ruler, measure the length (in centimeters) of the cells observed on the screen and enter the values on Table 3. Calculate the average length for each condition.


Table 3: Yeast Cell Length

Yeast Type Cell Length: no HU (cm) Cell Length: with HU (cm)

Wild type

Average length: Average length:

Mutant #1


Mutant #2


How does the average cell length compare between the wild type and mutants (both untreated HU- and treated HU+ conditions)?


4. Using Table 4, classify mutant #1 and #2 as either checkpoint or DNA repair mutant.

Mutant #1:____________________________________________________

Mutant #2:_____________________________________________________

Table 4: Yeast Characteristics for Mutant Identification

Yeast Type Cell CharacteristicsLight Microscope

Cell CharacteristicsFluorescent Scope

Wild type HU- Medium length cellsAll cell cycle stages present

Few Rad22-gfp fociEqual DNA distribution between cells

Wild type HU+ Longer cellsNo or few dividing cells

Increased Rad22-gfp fociRad22 localized in the nucleusEqual DNA distribution between cells

Checkpoint mutant HU- Wild type appearance Wild type appearance

Checkpoint mutant HU+ Shorter cellsMany dividing cells

Increased Rad22-gfp fociDNA unequally distributed between cells

DNA repair mutant HU- Wild type appearance Wild type appearance

DNA repair mutant HU+Longer cellsNo or few dividing cells (similar to wild type + HU)

Presence or absence of increased Rad22-gfp indicates location of mutation in repair pathway

5. Prepare a slide set of the mutant yeast you created with UV radiation and located through replica screening. Swirl the flask to mix the cells, pipette 5ul of culture onto a glass slide, and add a coverslip. Make an HU- untreated slide and a HU+ treated slide.

6. Classify your mutants by categorizing 25 cells according to the characteristics in Table 4. Record your classifications in Table 5.

Table 5: Classification of Mutant Yeast, Light Microscopy

Mutant # Average cell length:No HU (cm)

Average cell length: With HU (cm)

Mutant type:DNA repair or checkpoint?


Part B: Examination of DNA and DNA-repair proteins using a fluorescence microscope1. Examine the wild type and mutant slide sets under the fluorescent microscope. Compare and

contrast wild type and mutant yeast, both treated HU+ and untreated HU- conditions.2. Using the DAPI filter, observe the DNA distribution between daughter cells.3. Using the GFP filter, observe the abundance of Rad22-gfp protein in the nucleus.4. Record your observations in Table 6 and classify each mutant as DNA repair or checkpoint.

Table 6: Classification of Mutant Yeast , Fluorescence Microscopy

Yeast type DNA distribution(equal or unequal)

RelativeGFP production(compared to HU- for each yeast type)

Mutant type: DNA repair or checkpoint?

Wild type HU- reference N/A

Wild type HU+ N/A

Mutant #1 HU- reference

Mutant #1 HU+

Mutant #2 HU- reference

Mutant #2 HU+


What is occurring at the molecular level when you are able to observe Rad22-gfp?

Propose an explanation for the unequal DNA distribution in some of the daughter cells after the yeast were exposed to HU.

Propose an explanation for the increased abundance of Rad22-gfp in some of the daughter cells after the yeast were exposed to HU.


Documents

· Web viewThe 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