Download Lab 7 Mitosis Meiosis

Cell Division Lab: Mitosis and Meiosis
Investigation 7
AP Biology
Essential Question: How do eukaryotic cells divide to produce genetically identical cells or to
produce gametes with half the normal DNA?
Objectives:
 To describe the events in the cell cycle and how these events are controlled.
 To explain how DNA is transmitted to the next generation via mitosis.
 To explain how DNA is transmitted to the next generation via meiosis followed by
fertilization.
 To understand how meiosis and crossing over leads to increased genetic diversity which is
necessary for evolution.
Background:
All new cells come from previously existing cells. New cells are formed by the process of
cell division, which involves both division of the cell’s nucleus (karyokinesis) and division of the
cytoplasm (cytokinesis). There are two types of nuclear division: mitosis and meiosis.
Mitosis typically results in new somatic (body) cells. Formation of an adult organism from a
fertilized egg, asexual reproduction, regeneration, and maintenance or repair of body parts are
accomplished through mitotic cell division. Where does one find cells undergoing mitosis? Plants and
animals differ in this respect. In higher plants the process of forming new cells is restricted to
special growth regions called meristems. These regions usually occur at the tips of stems or roots.
In animals, cell division occurs anywhere new cells are formed or as new cells replace old ones.
However, some tissues in both plants and animals rarely divide once the organism is mature.
To study the stages of mitosis, you need to look for tissues where there are many cells in
the process of mitosis. This restricts your search to the tips of growing plants, such as the onion
root tip, or, in the case of animals, to developing embryos, such as the whitefish blastula. Roots
consist of different regions (see Figure below). The root cap functions in protection. The apical
meristem is the region that contains the highest percentage of cells undergoing mitosis. The region
of elongation is the area in which growth occurs. The region of maturation is where root hairs
develop and where cells differentiate to become xylem, phloem and other tissues.
To estimate the relative length of time that a cell spends in the various stages of cell
replication, you will examine the meristematic region of a prepared slide of the onion root tip. The
meristematic regions are the areas in plants undergoing active cell growth. The length of the cell
cycle is approximately 24 hours for cells in actively dividing onion root tips.
It is hard to imagine that you can estimate how much time a cell spends in each phase of cell
replication from a slide of dead cells. Yet this is precisely what you will do in this part of the lab.
Since you are working with a prepared slide, you cannot get any information about how long it takes
a cell to divide. What you can determine is how many cells are in each phase. From this, you can
infer the percent of time each cell spends in each phase.
The cell cycle is divided into three parts: interphase, mitosis and cytokinesis. Interphase
is separated into three functionally distinct stages. In the first growth phase (G1), the cell grows
and prepares to duplicate its DNA. In synthesis (S), the chromosomes are replicated; this stage is
between G1 and G2. In G2, the cell prepares to divide. In most cases, mitosis is followed by
cytokinesis, when the cytoplasm divides and organelles separate into daughter cells.
Cell division is tightly controlled by complexes made of several specific proteins. These
complexes contain enzymes called cyclin-dependent kinases (CDKs), which turn on or off the various
processes that take place during cell division. CDK partners with a family of proteins called cyclins.
One such complex is mitosis-promoting factor (MPF), sometimes called maturation-promoting
factor, which contains cyclin A or B and CDK. CDK is activated when it is bound to cyclin,
interacting with various other proteins that, in this case, allow the cell to proceed from G2 into
mitosis. The levels of cyclin change during the cell cycle. In most cases, cytokinesis follows
mitosis.
Different CDKs are produced during the phases of mitosis. The cyclins determine which
processes in cell division are turned on or off and in what order by CDK. As each cyclin is turned on
or off, CDK causes the cell to move through the stages in the cell cycle.
Cyclins and CDKs do not allow the cell to progress through its cycle automatically. There
are three checkpoints a cell must pass through: the G1 checkpoint, G2 checkpoint and the Mspindle checkpoint. At each of the checkpoints, the cell checks that is has completed all of the
tasks needed and is ready to proceed to the next step in its cycle. Cells pass the G1 check point
when they are stimulated by appropriate external growth factors; for example, platelet-derived
growth factor (PDGF) stimulates cell near a wound to divide so that they can repair the injury. The
G2 checkpoint checks for damage after DNA is replicated, and if there is damage, it prevents the
cell from going into mitosis. The M-spindle (metaphase) checkpoint assures that the mitotic
spindles or microtubules are properly attached to the kinetochores (anchor sites on the
chromosomes). If the spindles are not anchored properly, the cell does not continue on through
mitosis. The cell cycle is regulated very precisely. Mutations in cell cycle genes that interfere with
proper cell cycle control are found very often in cancer cells.
Sordaria fimicola is an ascomycete fungus that can be used to demonstrate the results of
crossing over during meiosis. Sordaria is a haploid organism for most of its life cycle. It becomes
diploid only when the fusion of the mycelia (very small filaments) of two different strains results in
the fusion of the two different types of haploid nuclei to form a diploid nucleus. The diploid nucleus
must then undergo meiosis to resume its haploid state.
Meiosis, followed by mitosis, in Sordaria results in the formation of eight haploid
ascospores contained within a sac called an ascus (plural, asci). Many asci are contained within a
fruiting body called a perithecium. When ascospores are mature the ascus ruptures, releasing the
ascospores. Each ascospore can develop into a new haploid fungus..
To observe crossing over in Sordaria, one must make hybrids between wild-type and mutant
strains of Sordaria. Wild-type (+) Sordaria have black ascospores. One mutant strain has tan
spores (tn). When mycelia of these two different strains come together and undergo meiosis, the
asci that develop will contain four black ascospores and four tan ascospores. The arrangement of
the spores directly reflects whether or not crossing over has occurred. In Figure 2, no crossing
over has occurred. Figure 3 shows the results of crossing over between the centromere of the
chromosome and the gene for ascospore color.
Fig. 2: Formation of Non-crossover Asci
Two homologous chromosomes line up at metaphase I of meiosis. The two chromatids of one
chromosome each carry the gene for tan spore color (tn) and the two chromatids of the other
chromosome carry the gene for wild-type spore color (+).
The first meiotic division (MI) results in two cells each containing just one type of spore
color gene (either tan or wild-type). Therefore, segregation of these genes has occurred at the
first meiotic division (MI). The second meiotic division (MII) results in four cells, each with the
haploid number of chromosomes (lN). A mitotic division simply duplicates these cells, resulting in 8
spores. They are arranged in the 4:4 pattern.
Fig. 3: Formation of Crossover Asci
In this example, crossing over has occurred in the region between the gene for spore color
and the centromere. The homologous chromosomes separate during meiosis I. This time, the MI
results in two cells, each containing both genes (1 tan, 1 wild-type); therefore, the genes for spore
color have not yet segregated. Meiosis II (MII) results in segregation of the two types of genes
for spore color. A mitotic division results in 8 spores arranged in the 2:2:2:2 or 2:4:2 pattern.
Any one of these spore arrangements would indicate that crossing over has occurred
between the gene for spore coat color and the centromere.
Two strains of Sordaria (wild-type and tan mutant) have been inoculated on a plate of agar.
Where the mycelia of the two strains meet (Figure 4), fruiting bodies called perithecia develop.
Meiosis occurs within the perithecia during the formation of asci. A slide has been prepared of
some perithecia (the black dots in figure 4).
Fig. 4
The frequency of crossing over appears to be governed largely by the distance between
genes, or in this case, between the gene for spore coat color and the centromere. The probability
of a crossover occurring between two particular genes on the same chromosome (linked genes)
increases as the distance between those genes becomes larger. The frequency of crossover,
therefore, appears to be directly proportional to the distance between genes.
A map unit is an arbitrary unit of measure used to describe relative distances between
linked genes. The number of map units between two genes or between a gene and the centromere is
equal to the percentage of recombinants. Customary units cannot be used because we cannot
directly visualize genes with the light microscope. However, due to the relationship between
distance and crossover frequency, we may use the map unit.
The frequency of crossing over appears to be governed largely by the distance between
genes, or in this case, between the gene for spore coat color and the centromere. The probability
of a crossover occurring between two particular genes on the same chromosome (linked genes)
increases as the distance between those genes becomes larger. The frequency of crossover,
therefore, appears to be directly proportional to the distance between genes.
A map unit is an arbitrary unit of measure used to describe relative distances between linked
genes. The number of map units between two genes or between a gene and the centromere is equal
to the percentage of recombinants. Customary units cannot be used because we cannot directly
visualize genes with the light microscope. However, due to the relationship between distance and
crossover frequency, we may use the map unit.
The Investigation
This investigation consists of four parts. In Part 1, you will observe cells under the microscope and
draw them. You will draw schematics and use them to guide you. In Part 2, you will determine the
effects of the environment on mitosis. You will compare observed/control values to experimental
values, using the chi square test. Note: you will write a paper on cancer and the cell cycle in the
near future. In Part 3, you will model meiosis with pop-beads. In Part 4, you will observe crossing
over in Sordaria. You will determine the percentage of crossing over (recombination frequency) and
then map units for the gene that controls asci color.
General Safety Precautions:
You must be careful when preparing specimens for viewing under the compound microscope. Always
cover the cover slip with a scientific cleaning wipe, such as a Kimwipe, and press down using a pencil
eraser.
You should wear safety goggles or glasses and disposable gloves when handling chemicals and razor
blades. All materials should be disposed of properly as per your teacher’s instructions.
Vocabulary – To be used in your lab introduction. Underline ALL terms. (17 Total)
Centromere
Ascospore
CDks
checkpoint
Ascus
Recombination
root cap
cell cycle
Recombination frequency
Mutant
region of elongation
cyclins
Perithecium
Apical meristem
region of maturation
MPF
Cytotoxic
Prelab: Print or write results from these activities neatly into your journal.
1. Go http://www.phschool.com/science/biology_place/labbench/lab3/intro.html
2. Read through the Mitosis I Introduction, Key Concepts I, and Design of the Experiment I.
Complete the Analysis of Results I and LabQuiz I.
3. Go http://www.phschool.com/science/biology_place/labbench/lab3/intro.html
4. Read through the Meiosis II Intro, Key Concepts II and Design of the Experiment II.
Complete the Analysis of Results-II and LabQuiz II.
PreLab
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Questions:
Where do new cells come from?
Distinguish between the terms karyokinesis and cytokinesis.
What type of cell is formed by meiosis?
What type of cell is formed via mitosis? Describe four functions of mitotic division.
Where does one find cells undergoing mitosis in a plant? In an animal?
Where does mitosis occur in a plant root?
Name the three zones of a root; briefly describe the function of each.
What type of fungus is Sodaria fimicola?
Why are there 8 spores in an ascus?
Summarize the key points of the Sordaria life cycle and crossing over. Use the background
information provided below or found in your textbook and online.
Day 1 Part 1: Viewing Cells in Mitosis
Part 1



Materials:
Prepared onion root tip slide
Microscope
Colored Pencils/Camera
Part 1 PROCEDURES:
1. Using either your textbook or the internet, draw a plant cell in each mitotic phase in Table 1
(Schematic Drawing). We are drawing plant cells because we will be observing onion root tips.
Label centromere, spindle fibers, asters, chromatin, chromosomes and chromatids where
appropriate, nucleus and cytoplasm.
2. Provide a description of the major events taking place during each mitotic phase (Description).
3. Obtain a prepared slide of an onion root tip. Use 40X, then 100X magnification on a compound
microscope to locate the meristematic region of the onion root tip. Study individual cells at
400X. Identify one cell that clearly represents each phase of mitosis; sketch or photograph and
label the cells in Table 3.1 (Drawing or Photograph from Prepared Slide). If sketching, your
sketch should be a careful drawing of what you see under the microscope.
Table 1.1: Mitosis
Mitotic Phase
Interphase
Prophase
Metaphase
Anaphase
Telophase
Schematic Drawing w/4
chromosomes-labeled
(from your textbook)
Description
Drawing or Photograph
from Prepared Slidelabeled
(Onion Root)
Day 1 Part 2: Effects of Environment on Mitosis
Procedure for Control Groups:
1. Observe the prepared onion root tip cells at high magnification (400x).
2. Within the field of view, count the cells in each phase.
3. Collect the class data and calculate the mean and standard deviation for each phase.
4. Calculate the percentage of cells in each phase.
5. Graph the class data to illustrate a comparison between the number of cells in interphase
vs. mitosis in onion root tip cells.
Table 2.1: Onion Root Tip Cell Phase Data: Your Lab Group (Totals)
Tip
Number of Cells
Treatment
Interphase
Mitosis
Control
Total
Percentage in Interphase:
Percentage in Mitosis:
Table 2.2: Onion Root Tip Cell Phase Data: Entire Class Data (Totals)
Tip
Number of Cells
Treatment
Interphase
Mitosis
Control
Mean number of cells in Interphase ____________
Mean number of cells in Mitosis ______________
Percentage in Interphase:
Percentage in Mitosis:
Standard Deviation for each Phase:
Total
Procedure for Experimental Groups:
1. Choose either lectin or caffeine (choose only 1 concentration) as a treatment.
2. Form a hypothesis and null hypothesis for the effects of the treatment on the cells of
the onion root tip.
3. Calculate the chi square value for the data and either accept or fail to accept the null
hypothesis.
4. Generate a graph to illustrate the effects of treatment on onion root tip cells.
Hypothesis:
Null Hypothesis:
Results: (observed at 400 x)
Table 2.3: Onion Root Tip Cell Phase Data; Treatment group with Lectin or Caffeine
Tip
Number of Cells
Treatment
Interphase
Prophase
Metaphase
Anaphase/Telophase
Control
460
43
8
24
Lectin
417
117
51
41
1 mM
549
18
3
5
caffeine
5 mM
449
11
2
7
caffeine
10 mM
330
9
3
0
caffeine
100 mM
Cytotoxic ------------------------------
caffeine
Total
535
626
575
469
342
Note: To determine the total number of cells in mitosis, you will need to add up the total cells in
Prophase, Metaphase, Anaphase and Telophase. Also, be sure to show your work.
Part 2
1.
2.
3.
Analysis Questions:
What was the importance of collecting class data?
Was there a significant difference between the groups?
Did the fungal pathogen lectin or the caffeine increase the number of root tip cells in
mitosis?
4. What other experiments should you perform to confirm/verify your findings?
5. Does an increased number of cells in mitosis mean that these cells are dividing faster than
the cells in the roots with a lower number of cells in mitosis?
6. What other way could you determine how fast the rate of mitosis is occurring in root tips?
Part 3 Modeling Meiosis
Part 3




Materials:
Internet Access
Pop-Beads
White Board
Dry Erase Markers
Part 3 PROCEDURES:
1. Create your own model with the pop-beads. Your model should include 4 sets of homologous
chromosomes.
2. Create a video or set of pictures that illustrate the chromosomes (pop beads) in each phase
in meiosis.
Part 4: Crossing-Over in Sordaria
Part 4 Procedures:
1. Obtain a set of color photographs that show several “squished” perithecia that resulted
from a fusion of black wild type and tan mutant haploid mycelia. Alternatively, you may be
asked to view prepared slides or prepare a wet mount. Each perithecium contains many acsi;
each ascus contains 8 ascospores.
2. Your job is to count at least 50 hybrid asci, determining the #asci with non-crossover
(4:4) and crossover (2:4:2 or 2:2:2:2) patterns within the asci. Enter your data in Table 4.1.
Calculations for Table 4.1:
 % Crossover = # Crossover asci divided by total #asci X 100
 Map Distance = % Crossover asci divided by 2 (to account for the effects of mitosis
– only half of the spores in each ascus are the result of meiosis)
Table 4.1: % Crossing Over and Map Units
# Non-Crossover
# Crossover Asci
Total # Asci
Asci
(2:4:2 or 2:2:2:2)
(4:4)
% Asci
showing
crossover
Gene to
Centromere
Distance
(Calculated as %
Asci showing
crossover ÷ 2
(Centimorgans))
Part 4 ANALYSIS QUESTIONS.
1. Why did you divide the percentage of asci showing crossover by 2?
2. Do you think the cell cycle described for mitosis could be applied to meiosis as well?
Explain.