TEACHER/TECHNICIAN GUIDE

Prize Winning Yeast - A practical exploring the control of the cell cycle

Contents

Background Information on the practical

Schizosacchromysces pombe: a fission yeast

Detailed description of the S.pombe cell cycle

Objectives

Cell Cycle mutants used in this practical

Extension Activities

References and Websites

Risk Assessment

Preparation of materials

Answers to Student Activity Sheet 1

Background Information

Schizosaccharomyces pombe: a fission yeast

In 1893, Lindner discovered a yeast in East African millet beer, locally called pombe and he named the yeast Schizosaccharomyces pombe. S. pombe is an example of a fission yeast. Yeast cells are single celled eukaryotes that undergo a cell division cycle like mammalians cells. They are haploid, that is, they each carry one copy of each chromosome.

The student guide contains more background information on S. pombe. One particularly confusing issue with the S. pombe cell cycle is that cell division does not occur directly after mitosis (as is seen the traditional mammalian cell cycle). Rather cytokinesis and daughter cell separation occur in G1 and S phases alongside all the other activities of these phases. It can be seen as an overlap from one cell cycle to the next. See overhead 1 for diagram summarising the mammalian cell cycle. This can be used to facilitate comparison of the cell cycles. Note that the nuclear activities at each stage of cell cycle are the same between yeast and mammalian cells.

Picture 1 – The S. pombe cell cycle

It is possible to predict where S. pombe cells are in the cell cycle simply by looking out for certain landmarks, as is shown below.

Detailed description of the S. pombe cell cycle.

/ G1 Phase: Mitosis has been completed and each cell contains two identical haploid nuclei. Mammalian cells would have divided directly after mitosis. However fission yeast start a new round of the cell cycle before the cells have separated. Therefore during G1 phase cytokinesis starts. Cytokinesis is where the cytoplasm is divided in two (with one nucleus on each side of the divide). In fission yeast this happens by the formation of a septum centrally across the cell.
/ S phase: In fission yeast two things are happening during S phase. First, the final separation into two daughter cells is occurring. Therefore cells in S phase can be identified by the presence of a septum. Second, DNA synthesis is occurring. In each of the haploid daughter cells an exact copy of each chromosome is made. By the end of S phase the daughter cells are diploid, that is each of them contains two copies of each chromosome.


/ G2 phase: by the time G2 phase begins, complete separation of the cells will have occurred and they will each be diploid. During G2 the cell elongates until it has reached a big enough size, such that there is sufficient cytoplasm and molecular machinery to let the cell enter and complete mitosis. During G2 there are also various checks, such as checking that DNA synthesis was successfully completed.
/ M phase: The cell contains two identical copies of each chromosome (diploid). Mitosis is the process through which these chromosomes are divided equally into two new nuclei. Mitosis has 4 main stages: Prophase, metaphase, anaphase and telophase. These are described on a separate poster.

Objectives

This practical has four main parts:

1)  Growing two mutant yeast strains at different temperatures.

2)  Observing the yeast using a microscope and recording observations.

3)  Measuring the yeast using a graticule and constructing a bar chart.

4)  Drawing conclusions in relation to loss of cell cycle control for each of the mutant yeast.

Cell cycle mutants used in this practical

The key to recognising these mutants is the fact that the length of S. pombe is related to its stage in the cell cycle. Therefore cells with defective cell cycle control are abnormal sizes.

Line 1 is called wee 1ts (strain wee 1.50, ts stands for temperature sensitive). Wee 1 protein normally acts to prevent entry into mitosis until the cell has reached the required size. Therefore cells lacking functional wee 1 protein exhibit accelerated entry into mitosis. They enter mitosis at a smaller size before full growth is complete – hence the name ‘wee 1’. The wee 1 mutant cells can still survive even through they divide at an earlier stage of G2 phase than normal (or wildtype) cells would. Wee 1 mutants can be recognised by the fact they are much smaller than normal cells.

Line 2 is called Cdc 25ts (strain cdc 25.22). Cdc 25 is a protein involved in initiating mitosis or M phase. Cells which lack the functional cdc 25 protein arrest in G2 phase before mitosis begins. Cells continue to grow but cannot enter mitosis, therefore the cells exhibit an elongated phenotype. Interestingly, checkpoints ensuring the completion of DNA synthesis act on cdc 25. When DNA damage or incomplete DNA synthesis is detected, cdc 25 is stopped from initiating mitosis. When the damaged DNA has been repaired, cdc 25 is then allowed to initiate mitosis.

Line 1 and Line 2 are temperature sensitive mutants. That means that when the cells are incubated at room temperature (~ 20 °C) the cell cycle occurs as normal, as if wee 1 and cdc 25 are functioning normally. However when they are grown at 35°C the wee 1 and cdc 25 proteins change conformation to an inactive form, therefore the yeast cells lose control of the cell cycle in specific ways. It should be noted that wee 1 cells grown at 20 °C will still look much smaller than cdc 25 cells also grown at 20°C. See the ‘Answers to pupils activity sheet 1’ for pictures of each strain.

References and Websites

S.pombe - http://www.teaching-biomed.man.ac.uk/ramsay/Homep.htm

The Cell Cycle - http://www.cellsalive.com/

SAPS - http://www-saps.plantsci.cam.ac.uk/

SSERC - http://www.sserc.org.uk/

Risk Assessment

Student: Risk Assessment

Location: Classroom

Sequence of Activity / Student Involvement / Risk to Student participant / Action required
Inoculation of EMM Broth / Use of sterile loops / None / Aseptic technique must be used at all times.
Transfer of yeast cells onto microscope slides and applying coverslip. / Removal of small amount of broth containing yeast cells
Applying coverslip / None
Cutting themselves / Aseptic technique must be used at all times.
Due care and attention must be used.
Accidental spill of yeast culture / Cleaning up spill / None / Clean up spill and disinfect area. Wash hands.


Technician: risk assessment

Location: Technicians workbase

Sequence of Activity / Technician Involvement / Risk to Technician / Action required
Weighing out EMM powder and agar / Use of EMM powder and agar / Inhalation of powder, allergy to powder. / Wear appropriate hand and face protection.
Opening Vial containing freeze dried yeast. / Breaking open vial / Cutting on glass / Follow protocol instructions ensuring hands are well protected before opening vial.
Autoclaving EMM broth and agar / Use of autoclave / Burning / Follow manufacturers instructions, have equipment serviced regularly. Allow media to cool before handling
Spreading yeast culture on plates / Using sterile loop to inoculate plates / None / Aseptic technique required at all times
Disposal of cultures and contaminated glassware. / Disposal of laboratory reagents and contaminated materials / None / All contaminated materials must be autoclaved and disposed of as appropriate.

Summary of Risks

Good laboratory and aseptic technique must be used at all times. The appropriate hand, body and face protection must be worn. Spills should be cleaned up and the area disinfected. All cultures and contaminated glassware must be disposed of appropriately. Please refer to the microtechniques manual for further information and practical information and protocols on aseptic technique.

Extension activities

1)  Growth the yeasts at a range of temperatures (e.g. from 20°C to 50°C) and determine the initial temperature at which the cell cycle is affected.

2)  Investigate the change in length of Line 2 cells over time (e.g. after 4 hrs, 8hrs, 16hrs, 20hrs, 24hrs and so on). Please note this will involve setting up several cultures at different times.

3)  Irradiate S.pombe non mutant cells with UV light to induce mutations and investigate the effect of using different filters on mutation rate (as determined by percentage of cells with abnormal morphologies or number of cells in S phase).

Preparation of materials

Approximately 4-5 days before the practical S. pombe, wee 1 mutant (Line 1) and cdc 25 mutant (Line 2) cultures must be streaked out on EMM agar plates from the slopes provided. This can be done by pupils or alternatively by a teacher/technician.

To make up the EMM liquid medium (for 10 sterile jars):

Materials

200 cm3 beaker

stirring rod

10 sterile jars

weighing boat

EMM powder

Distilled water

Marker pen

1.  Pour approximately 80cm3 into a beaker.

2.  Weigh out 3.2g EMM powder

3.  Place chemicals into the beaker and stir thoroughly with a stirring rod.

4.  Add enough water to make up to the 100cm3 mark on the beaker.

5.  Place 10cm3 of this medium into each of the sterile jars and label them.

6.  Autoclave at 121°C for 15 minutes.

To make up the EMM agar plates (For 6 plates):

Materials

200 cm3 beaker

stirring rod

glass bottle

weighing boat

EMM powder

Distilled water

Marker pen

agar

1.  Follow instructions 1 – 4 above.

2.  Pour into a glass bottle and label.

3.  Add 2g of agar.

4.  Mix and then place lid loosely on bottle.

5.  Autoclave for 15 minutes.

NB if this is done by pupils then it is advisable at step 4 to place 15cm3 of the solution into each of 6 McCartney bottles. Then add 0.3g of agar to each bottle. Autoclave.

Pouring the Agar

Materials

Glass bottles with sterile molten EMM agar

Disinfectant (type) and cloth

Labels

Sterile Petri dishes

Bunsen burner

1.  Wash hands.

2.  Clean area which you are going to work in with a cloth or tissue soaked in disinfectant.

3.  Label Petri dish (take care not to open them) with EMM agar and the date.

4.  When agar is cool enough to handle take off lid and flame the bottle in a cool Bunsen burner.

5.  Pour approximately 15cm3 agar into each Petri dish. Open the dishes as little as possible.

6.  Once agar has set it is ready to be inoculated with the yeast. If EMM agar is to be stored it must be kept at 4°C in the dark (for example, wrapped in tin foil).

Rehydration of Freeze-dried S.pombe mutants

Mark the glass with a file or other glass cutter in the area of the cotton bung. Wrap a paper towel several times around the marked area and break open. Using a sterile pipette add a few cm3 of EMM broth medium to the yeast cells and mix with a sterile loop. Transfer the S.pombe suspension to a prepare 10-20ml bottle of yeast medium and leave at room temperature out of direct light for a few days before using for inoculation.

Streaking an agar plate

Materials

Disinfectant and cloth

EMM plates

S. pombe suspension.

Sterile plastic inoculating loops

Sellotape

Marker pen

1.  Swab working area with disinfectant

2.  Label Petri disk with S. pombe strain and date

3.  Open your Petri dish and using a sterile inoculating loops streak out the yeast in the pattern shown below.

4.  Seal the plate using two small strips of sellotape.

See student materials for practical protocol.


ANSWERS TO PUPIL ACTIVITY SHEET 1

1)  Examine the slides of Line 1 and Line 2 cells grown at 20°C. Can you identify the different stages in the S. pombe cell cycle according to cell size and the presence of septa? Use picture 3 for help.

NB Sometimes even when grown at 20°C the cells in Line 1 look quite abnormal.

2)  Draw pictures below to show the appearance of the cells.

3)  Examine slides of Line 1 and Line 2 cells grown at 35°C. What do you notice? Draw your observations in the boxes below.

4)  Use an eye piece micrometer to measure the lengths of 10 cells from each line grown at both temperatures.

You will need to decide on some constants for your measurements. For example:

a)  The largest 10 cells on the slide will be measured.

b)  Cells with septa will be omitted from the measurements.

c)  Cells which are in the process of dividing (i.e. the two daughter cells are still attached) will be omitted from the measurements


Record your data in the table below.

Name / Growth Temp / Length (in grid units) / Average
Line 1 / 20°C / 2,3,3,2,3,2,2,2,2,2 / 2.3
Line 1 / 35°C / 2,1,1,3,1,1,2,2,1,1 / 1.5
Line 2 / 20°C / 3,2,4,4,3,5,3,3,5,3 / 3.5
Line 2 / 35°C / 12,11,12,15,12,13,12,13,12,11 / 12.3

5)  Construct a bar chat showing average cell length for Line 1 and Line 2 at 20°C and 35°C.

6)  Use your knowledge of the S.pombe cell cycle to detect which stage in the cell cycle is affected by each mutation and give evidence for your conclusion.

Line / Protein Affected / Stages Affected / Evidence
Line 1 / wee 1 / G2/M / The cells are abnormally small. They are not going through the full growth phase in G2 and are passing prematurely into M phase.
Line 2 / cdc 25 / G2 / The cells are abnormally large. They are getting blocked in G2 phase and are never passing into M phase.

9

December 2003

STUDENT GUIDE

Introduction

Schizosaccharomyces pombe: a fission yeast

Yeast cells are single celled eukaryotes that undergo a cell division cycle similar to mammalians cells. They are haploid, i.e. they each carry one copy of each chromosome. S. pombe is an example of a fission yeast.