Download Yeast cells, which are simple, single celled eukaryotes, undergo cell

December 2003
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 he called Schizosaccharomyces pombe
in East African millet beer, locally called 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
1
December 2003
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.
This cell has undergone S phase and complete separation of the two daughter cells has
occurred. It is at the start of G2 phase.
This cell has undergone elongation in G2 phase. However as we cannot visualise the
nuclei it could be at the end of G2 phase, in M phase or in G1 phase.
This cell is in S phase (DNA synthesis). In S. pombe the separation into two daughter
cells from the previous cell cycle also occurs in S phase. To do this the cell forms a
septum. Therefore the presence of a septum tells us the cell is in S phase.
This cell is at the end of S phase and complete separation of the daughter cells is nearly
complete.
2
December 2003
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.
Septum
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.
3
December 2003
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 (stain 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 DNA synthesis is complete 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.
4
December 2003
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
Inoculation of EMM
Broth
Student
Involvement
Use of sterile loops
Risk to Student
participant
None
Transfer of yeast
cells onto
microscope slides
and applying
coverslip.
Removal of small
amount of broth
containing yeast cells
None
Applying coverslip
Cutting themselves
Accidental spill of
yeast culture
Cleaning up spill
None
Action required
Aseptic technique
must be used at all
times.
Aseptic technique
must be used at all
times.
Due care and
attention must be
used.
Clean up spill and
disinfect area. Wash
hands.
Technician: risk assessment
Location: Technicians workbase
5
December 2003
Sequence of
Activity
Weighing out EMM
powder and agar
Technician
Involvement
Use of EMM powder
and agar
Risk to Technician
Action required
Inhalation of powder,
allergy to powder.
Opening Vial
containing freeze
dried yeast.
Breaking open vial
Cutting on glass
Autoclaving EMM
broth and agar
Use of autoclave
Burning
Spreading yeast
culture on plates
Disposal of cultures
and contaminated
glassware.
Using sterile loop to
inoculate plates
Disposal of
laboratory reagents
and contaminated
materials
None
Wear appropriate
hand and face
protection.
Follow protocol
instructions ensuring
hands are well
protected before
opening vial.
Follow manufacturers
instructions, have
equipment serviced
regularly. Allow
media to cool before
handling
Aseptic technique
required at all times
All contaminated
materials must be
autoclaved and
disposed of as
appropriate.
None
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).
6
December 2003
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
7
December 2003
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
8
December 2003
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.
1
5
4
2
3
See student materials for practical protocol.
9
December 2003
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 1l look quite abnormal.
2) Draw pictures below to show the appearance of the cells.
Line 1 - 20°C
Line 1 - 35°C
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.
Line 2 - 20°C
Line 2 - 35°C
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.
10
December 2003
Name
Growth
Length (in grid units)
Average
Temp
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.
14
Average (grid units)
12
wee 1 Room
Temperature
10
wee 1 35ºC
8
6
cdc 25 Room
Temperature
4
cdc 25 35ºC
2
0
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
Line 1
Protein
Stages
Affected
Affected
wee 1
G2/M
Evidence
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.
11
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, that is they each carry one
copy of each chromosome. S. pombe is an example of a fission yeast.
You may be more
familiar
with
Picture 1
the
budding
yeast,
Budding Yeast
Fission Yeast
Saccharomyces
cerevisiae.
See
Picture 1 to compare
the
apprearance
these
tow
of
yeasts
under a microscope.
S. pombe cell cycle
The goal of the cell cycle is to provide two daughter cells which are exactly
the same as the mother cell.
The S.pombe cell cycle is divided into the four same phases (G1, S, G2, M)
as the mammalian cell cycle and the nuclear activities at each stage are the
same in yeast as in mammalian cells. However the S.pombe yeast cell cell
separation occurs in G1/S phases rather than directly after mitosis.
See
picture 2.
1
December 2003
Picture 2
S.pombe grows by elongation, thus by measuring a cell, it’s position in the cell
cycle can be deduced. Other features can also help you determine this, as
illustrated below.
Picture 3
This cell has undergone S phase and complete separation of the two daughter cells has
occurred. It is at the start of G2 phase.
This cell has undergone elongation in G2 phase. However as we cannot visualise the
nuclei it could be at the end of G2 phase, in M phase or in G1 phase.
This cell is in S phase (DNA synthesis). In S. pombe the separation into two daughter
cells from the previous cell cycle also occurs in S phase. To do this the cell forms a
septum across the cell. Therefore the presence of a septum tells us the cell is in S phase.
This cell is at the end of S phase and complete separation of the daughter cells is nearly
complete.
Nobel prize-winning discovery
It is vital for all living eukaryotic organisms that the phases of the cell cycle are
precisely co-ordinated. They must occur in the correct order and each phase
must not start until the previous one is satisfactorily completed. If mistakes in
this co-ordination occur, chromosomes carrying essential code for the life of
2
December 2003
the organism may be changed. For example, chromosomes may be lost,
genes may be copied in the wrong order or they may not be divided equally
amongst each of the new daughter cells. These kinds of changes to
chromosomes are often seen in cancer cells, indicating that defective control
of the cell cycle can cause cells to become cancerous.
In 1975 a scientist called Paul Nurse and his colleagues carried out work
using S.pombe.
He found that the protein products of certain genes are
dedicated to ensuring that the cell cycle is carried out perfectly. That is, each
phase occurring in the correct order and all the chromosomes being copied
accurately once and only once. He named these genes cell division control
genes – or cdc genes. This ground breaking work and that of contemporaries
opened up a huge field in biological research. In 2001, Paul Nurse, Leland
Hartwell and Tim Hunt won the Nobel prize for Physiology or Medicine for
their discoveries relating to the cell cycle and the control of it.
Cancer in humans
It has now found that humans have genes similar to the S. pombe cdc genes,
they are called CDK (cyclin dependent kinases) genes. It has been shown
that faulty CDK genes can function as oncogenes (or cancer promoting
genes). Other genes, such as p53, prevent cancer by promoting cell cycle
arrest and cell death. In the future, biomedical scientists hope to use what
they know about cell cycle control to devise new therapies for cancer. Already
clinical trials are in progress using inhibitors of CDK protein.
Cell cycle mutants
In this practical two mutant strains of S.pombe are cultured at different
temperatures and the cells are examined under a microscope to determine
the effect of the environment on their growth and division. As the length of the
S.pombe cells is related to their stage in the cell cycle, yeast cells with
defective cell cycles are abnormal sizes.
3
December 2003
You will be looking at two particular lines of S.pombe: Line 1 has a mutation
in a gene which codes for a protein known as ‘wee 1’. Line 2 has a mutation
in a gene coding for a protein known as ‘cdc 25’.
Lines 1 and 2 are temperature sensitive mutants. The cell cycle occurs as
normal at room temperature. However, when the yeast is grown at 35°C the
proteins (wee1 and cdc25) coded for by the mutant genes change
conformation to inactive forms. The yeast cells therefore lose control of the
cell cycle in two different ways.
You are going to culture cells of both lines in liquid media at 20°c and 35°C.
You will then examine the cells under a microscope and measure the lengths
of the cells grown at each temperature. You will use your knowledge of the
cell cycle to determine which stage in the cell cycle is affected by each
mutation.
Sample Protocol
Materials needed by each person or group
Agar plates with mutant cultures of S. pombe (Line 1 and Line 2)
4 sterile screw topped jars containing 10 cm3 EMM
Bunsen burner
Sterile plastic Inoculating loops
Microscope (or share microscope between groups)
Eyepiece micrometer
Microscope slides and coverslips
Pen and labels
Disposal jars
Materials to be shared
water bath
disinfectant and paper towels
4
December 2003
Safety
Aseptic technique must be used at all times. 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.
1) You will be supplied with 2 plates containing cultures of two mutant S.
pombe lines, labelled Line 1 and Line 2. Line 1 is the ‘Wee 1’ line and
Line 2 is the ‘cdc 25 ts’ line.
2) Collect 4 sterile jars containing 10 cm3 EMM and label two jars ‘Line
1’and two jars ‘Line 2’. Don’t forget to put your name on them as well.
3) Inoculate each sterile jar with a single colony from the appropriate
plate. Use a different inoculating loop for each jar. Dispose of used
loops in the discard jar. You should now have two jars inoculated with
Line 1 and two jars inoculated with Line 2.
4) Label one set of jars (Line 1 and Line 2) as 20°C and the other set as
35°C.
5) Incubate (with occasional agitation) each set of jars at the appropriate
temperature overnight.
Note on incubation time.
The minimum incubation time is 5-6hrs and the maximum
incubation time is 24hrs. You can adapt this to suit your timetable.
The following day.
1) Collect all four jars from the incubators or water baths.
2) Label four microscope slides ‘Line 1-20°C’, ‘Line 2-20°C’, ‘Line 1-35°C’
and ‘Line 2-35°C’.
3) Using a disposable plastic pipette remove a small amount of yeast
culture from each of the four cultures and place one drop on the
appropriate glass microscope slide.
Remember to use a fresh
disposable plastic pipette for different cultures, to avoid contamination.
Sometimes clumps of yeast cells form, try to break these up using the end of the plastic
pipette. It can also help to give the culture a good swirl before carrying out instruction 3).
4) Carefully apply the coverslip using the “toothpick” method described
below. Try to avoid air bubbles.
5
December 2003
Toothpick method
1) Apply coverslip so that
one edge just touches the
edge of the drop of
culture.
2) Balance coverslip on a
toothpick.
3) Slowly lower the
coverslip using the
toothpick.
4) When the coverslip is
nearly flat, move the
toothpick to the edge of
the coverslip and finally
let the coverslip drop
onto the slide.
5) Use a microscope (at X200 or X400 magnification) to observe Line 1
and Line 2 incubated at 20°C and 35°C . The yeast are best observed
using a microscope with dark field however they can be viewed with
normal light.
6) Complete Student Activity Sheet 1 to record and analyse your results
6
December 2003
STUDENT ACTIVITY SHEET 1
7) 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 1l look quite abnormal.
8) Draw pictures below to show the appearance of the cells.
Line 1 – 20°C
Line 2 – 20°C
9) Examine slides of Line 1 and Line 2 cells grown at 35°C. What do you
notice? Draw your observations in the boxes below.
Line 1 – 35°C
Line – 35°C
10) 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:
d) The largest 10 cells on the slide will be measured.
e) Cells with septa will be omitted from the measurements.
f) Cells which are in the process of dividing (i.e. the two daughter cells
are still attached) will be omitted from the measurements
7
December 2003
Record your data in the table below.
Name
Growth
Temp.
Lengths (in grid units)
Average
Length (in
grid units)
Line 1
20°C
Line 1
35°C
Line 2
20°C
Line 2
35°C
11) Construct a bar chat showing average cell length for Line 1 and Line 2
at 20°C and 35°C.
8
December 2003
12) 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
Stage
Affected
Affected
Evidence
Line 1
Line 2
9