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GENERAL BIOLOGY LAB 1 (BSC1010L)
Lab #7: The Cell Cycle, Mitosis, Meiosis and Gametogenesis ______________________________________________________________________________
OBJECTIVES:
• Understand the major events involved in the cell cycle.
• Learn about the process of cellular division in plant and animal cells.
• Compare and contrast mitosis and meiosis.
• Understand the difference between male and female gametogenesis.
• Learn how to examine a karyogram.
• Perform karyotype analysis.
______________________________________________________________________________
INTRODUCTION:
The Cell Cycle
All eukaryotic cells undergo a series of growth and division events, collectively referred to as
the cell cycle (Fig. 1). The duration of the cell cycle is specific to cell type and organism. In
general, the cell cycle consists of three main phases: Interphase, Mitosis (M) and Cytokinesis
(C). The first stage, Interphase, is considered the non-dividing or growth portion is subdivided
into Gap 1 (G1), Synthesis (S), and Gap 2 (G2), of which G1 and G2 are the main growth
stages. Specifically, during G1 (the normal state of a cell), the cell grows and generates the
enzymes necessary for DNA replication that takes place during the S phase. In G2, the cell
synthesizes proteins, carbohydrates and lipids, which all function to increase the cell’s size, and
the chromosomes prepare to condense in preparation for the M phase.
Question:
Interphase is sometimes referred to as a “resting stage.” Why is this inaccurate?
The cell cycle is controlled by a series of checkpoints (Fig. 1), namely the G1/S, G2/M
and spindle checkpoints. The G1/S checkpoint, determines if the cell should continue into the S
phase or if it should enter a resting state (G0 = Gap 0 phase), which is important for cell types
that divide infrequently and/or cells that are terminally differentiated (e.g. nerve cells). This
checkpoint is followed by the G2/M checkpoint which serves as a control mechanism to prevent
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damaged cells from entering the M phase. Once the cells are committed to mitosis, the role of the
spindle checkpoint is to ensure that all chromosomes are attached to the mitotic spindle during
metaphase; if any chromosome is not attached, the cell will not be able to proceed into anaphase.
In addition, DNA damage checkpoints located in G1, S and G2 ensure that DNA is not damaged
before allowing the cell to proceed to mitosis. For example, the p53 protein, which plays a key
role in the G1 checkpoint, monitors the integrity of DNA during this stage. If the DNA is healthy
(i.e., no mutations) p53 will allow the cell to progress onwards through the cell cycle. On the
other hand, if p53 detects DNA damage, then it will arrest the cell in G1 either for repair or for
destruction (apoptosis = programmed cell death). If any of these checkpoints are nonfunctional
or mutated, control of the cell cycle is lost and cancer develops.
Figure 1. The cell cycle and its associated checkpoints
G2/M Checkpoint
Spindle Checkpoint
G1/S Checkpoint
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Questions:
a. How might you use the knowledge of the cell cycle checkpoints to prevent, diagnose, and
treat cancer?
b. What problems may occur as a result of having a mutated p53 protein?
Cellular Division: Mitosis vs. Meiosis
The genetic material (DNA) of all eukaryotic organisms is housed within the cell’s
nucleus and is passed on from generation to generation. While a cell is in interphase, the DNA
exists in an extended form called chromatin (Fig. 2) that repeatedly folds on top of itself,
condensing into visible chromosomes when the cell is ready to divide (i.e., entering the M phase
of the cell cycle). In somatic (body) cells, chromosomes exist in pairs and are called homologous
chromosomes. Each homologue within the pair is referred to as a sister chromatid and is joined
to the other by the centromere (Fig. 3). In eukaryotic organisms, the number of chromosomes
present differs between species but most eukaryotes are diploid (2n), meaning they have 2 pairs
of chromosomes. Chromosome number differs between organisms (Table 1), for example human
cells possess a total of 46 chromosomes (23 pairs), while canine cells possess a much larger
number (39 pairs).
Figure 2. Cell as it appears during Interphase Figure 3. A pair of sister chromatids
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Table 1. Chromosome numbers vary across species
Eukaryotic cells, depending on the type (somatic vs. germ cells), divide either by mitosis
or meiosis. Mitosis is the process in which a diploid parental cell is divided into 2 identical
daughter cells, also diploid in number. In contrast, meiosis involves the division of a diploid
parental cell into 4 daughter cells, all of which are haploid (n) in number. Mitosis occurs in
somatic cells, which are all cells of the body excluding the reproductive cells (eggs and sperm).
Meiosis, on the other hand, takes place only in the germ cells, i.e., cells of the reproductive
organs (testes and ovaries).
Mitosis (Fig. 4) is a nuclear event comprised of 4 stages, Prophase, Metaphase, Anaphase
and Telophase (although some authors describe Prometaphase as a distinct phase). Usually
following nuclear division, the 2 newly generated daughter cells are separated from each other
through the process of cytokinesis (division of the cytoplasm). During cytokinesis animal cells
form a cleavage furrow or indentation on the periphery of the cell that pulls the plasma
membrane inward, dividing the cell into two parts. Plant cells, in contrast, are unable to divide
using the cleavage furrow since they possess a rigid cell wall. Instead, they generate a cell plate
at the center of the cell that grows outward to split the cell into two.
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Figure 4. Mitosis
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Meiosis, on the other hand, occurs only in germ cells, i.e., those cells destined to become
gametes. This process is referred to as a reduction division since the 4 daughter cells generated
from the division of the diploid parental cell are haploid. The stages of Meiosis I are Prophase I,
Metaphase I, Anaphase I and Telophase I (Fig. 5) and of Meiosis II are Prophase II,
Metaphase II, Anaphase II and Telophase II (Fig. 6). Meiosis I involves the separation of
homologous pairs of chromosomes which are further separated into sister chromatids during
Meiosis II.
Figure 5. Meiosis I
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Figure 6. Meiosis II
During Prophase I of Meiosis, two pairs of homologous chromosomes form a tetrad through
synapsis and exchange genetic material via the process of crossing over (Fig. 7). In this
process, the genetic material is neither gained nor lost. Instead, new combinations of alleles arise,
thereby increasing genetic variation.
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Figure 7. Crossing over between homologous pairs of chromosomes
In today’s lab, you will examine the cell cycle, Mitosis and Meiosis. You will then
consider the role of the different phases of the cell cycle to understand the significance of each
step in the production of healthy cells and the possible consequences of mistakes during cell
division. Finally, you will learn how to examine karyotypes, which are used to determine the
number of chromosomes in a species as well as for the diagnoses of birth defects and genetic
abnormalities.
TASK 1 - Cycling Through the Cell Cycle
A) Identify the Stages of Mitosis
1. Examine a prepared slide of the whitefish blastula on high power.
2. Complete Table 2, making sure to draw examples of each phase of mitosis.
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Table 2:
Stage of Mitosis Description of Events Drawings of Stages
Prophase
Metaphase
Anaphase
Telophase
Questions:
a. Why are cells from a blastula used to examine mitosis?
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b. How fast do you think cells divide when an embryo is forming compared to the normal
growth of an animal?
c. How does cytokinesis differ between plant and animal cells?
B) Onion Root Tip Preparation
1. Using a scalpel cut the terminal 4mm of an onion root tip and add it to a small tube
containing 100µL of Carnoy's fixative.
2. Place the tube in a 60°C water-bath for 15 min to soften the tissue.
3. After 15 min, remove the onion tip from the fixative with forceps. Rinse the tip a 2-3
times with an ice cold 70% ethanol solution to remove any residual acetic acid from the
fixative. Note: Acetic acid reduces the ability to stain the chromosomes.
4. Place the root tip on a clean microscope slide and add a drop of Hydrochloric acid (HCl).
Using a dissecting microscope remove the very end of the tip. Keep this portion and
discard the remaining tissue.
5. With a dissecting needle, attempt to macerate/crush the tissue into small pieces.
6. Add one drop of Aceto-orcein stain to the crushed tissue.
7. Gently warm the slide by passing it over an ethanol lamp. DO NOT BOIL!!! Heating the
slide will speed up the staining process and allow some of the HCl in the stain to soften
the tissue.
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8. Allow the slide to sit for 1 min to cool down. In the meantime, smear a small amount of
Mayer's albumen onto a coverslip and allow it to dry. Make sure to set the coverslip face
up on your table so that you will know which side contains the albumen.
9. Place the slide on a piece of paper towel. Using forceps lower the coverslip (albumen side
down) over the stained tissue.
10. Place the end of the paper towel over the coverslip and, with your thumb, press down
onto the coverslip as hard as you can (but not so hard that the slide breaks). The act of
squashing separates the cells from each other, making the chromosomes more visible.
C) Time for Cellular Replication
1. Using your prepared onion root tip slide, count the number of cells in each phase of the
cell cycle (i.e., interphase and each stage of mitosis) in the high power field of view.
Repeat 3 times for an approximate total of 100 cells and record your results in Table 3.
2. Assuming that an onion root tip cell takes 14 hours (840 minutes) to complete the cell
cycle, the time that an onion cell spends in each stage of the cell cycle can be calculated
using the following formula:
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Time for each stage = Number of cells at each stage x 840 minutes
Total number of cells counted
Table 3:
Stage of Cell
Cycle
Number of Cells Time Spent in Each Stage
FOV 1 FOV 2 FOV 3 FOV 4 Total
Interphase
Prophase
Metaphase
Anaphase
Telophase
TASK 2 - Effect of Colchicine on Mitosis
Colchicine, a product of the plant Colchicum autumnale (common name = Meadow
saffron), is used in the treatment of gout, cirrhosis, and psoriasis, among other disorders (Ben-
Chetrit and Levy, 1998). During metaphase, chromosomes attach to the mitotic spindle via their
kinetochore and oscillate at the equatorial region under high tension. By interacting with tubulin,
a component of the spindle fibers, colchicine decreases this tension, suspending the
chromosomes in metaphase (Jordan and Wilson, 2004).
Develop a Hypothesis:
Based on the information above, propose null and alternate hypotheses about the stages of
mitosis that you would expect to see in cells treated with colchicine. Write your hypotheses (Ho
and Ha) below.
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Question:
Given colchicine’s properties, could this compound be used to treat cancer? Explain.
______________________________________________________________________________
TASK 3 – Meiosis and Gametogenesis
Gametes (sperm and eggs) are haploid reproductive cells that are formed by the process
of gametogenesis. In mammals and many other vertebrates, gametes and gametogenesis differ
between males and females; males produce sperm through the process of spermatogenesis (Fig.
8) while females produce eggs via oogenesis (Fig. 9).
Sperm is produced in the seminiferous tubules of the testes. Within the seminiferous
tubules, spermatogonia constantly replicate mitotically throughout the life cycle of males. Some
of the spermatogonia move inward towards the lumen of the tubule and begin meiosis. At this
point, they are called primary spermatocytes. Meiosis I of a primary spermatocyte produces
two secondary spermatocytes, each with a haploid set of double-stranded chromosomes.
Meiosis II separates the strands of each chromosome and produces two haploid spermatids that
mature and differentiate into sperm cells via spermiogenesis.
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Figure 8. Spermatogenesis
In females, oogenesis occurs in the oocytes of the ovaries. Unlike spermatogonia,
oocytes are not produced continuously. Oogonia, which are produced during early fetal
development, reproduce mitotically to produce primary oocytes. In humans, the ovaries of a
newborn female contain all the primary oocytes that she will ever have. At birth, primary oocytes
begin meiosis I, but are arrested in prophase I. At puberty, circulating hormones stimulate
growth of the primary oocytes in the follicles (surrounding tissue) each month. Just before
ovulation, the oocyte completes meiosis I producing a Graafian follicle which contains the
haploid secondary oocyte. Meiosis II proceeds but is not completed until fertilization occurs.
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Figure 9. Oogenesis
Questions:
1. Why do gametes have only half the number of chromosomes as the original parent cell?
Why is this important?
2. Would evolution occur without the events of meiosis and sexual reproduction? Why or
why not?
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Procedure:
1. Examine prepared slides of sperm from humans, rats, and guinea pigs and draw what
you see in the space provided below. How do the sperm from the three species
compare?
Magnification: _________
Magnification: _________
Magnification: _________
2. Examine a cross section of a monkey’s seminiferous tubules and draw what you see
in the space provided below. Locate the spermatogonia, primary spermatocytes,
secondary spermatocytes, spermatids and mature sperm and label each of these cells
on your drawing.
Magnification: _________
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3. Examine a cross section of cat ovary and draw what you see in the space provided
below. Locate and label the developing follicle with the egg inside on your drawing.
Magnification: _________
4. Examine the slide of a mature follicle (Graafian follicle) and draw what you see in the
space provided below.
Magnification: _________
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5. Compare Mitosis and Meiosis in Table 4:
Table 4:
Mitosis Meiosis
Purpose of process
Location
Number of cells
generated per cycle
Number of nuclear
divisions per cycle
Ploidy (n or 2n) of
daughter cells
Daughter cells genetically
identical to parent?
Pairing of homologues
Occurrence of crossing
over
Questions:
1. Why is meiosis referred to as reduction division?
2. If a species has 24 chromosomes in the nucleus prior to meiosis, what number will
each cell have after meiosis is complete?
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3. How do the sizes of the oocytes differ as they move from the follicle stage towards
the mature Graafian follicle?
4. How do sperm and eggs differ in size? (Hint: consider size and the quantity of each
gamete). Explain a possible reason for these differences.
5. What would happen if females produced 100’s or 1000’s of eggs during each cycle?
What if males were born with a limited number of sperm?
______________________________________________________________________________
TASK 4 - Karyotype Analysis
Karyotyping is the process by which scientists microscopically visualize the complete set of
chromosomes in an organism to detect any possible chromosomal abnormalities such as
deletions, translocations or the insertion of extra copies. Karyotype analysis is performed when
the chromosomes are highly condensed, i.e. in metaphase (halted in this phase with the addition
of colichicine). A normal human karyotype should consist of 22 autosomal pairs, listed from
largest (chromosome 1) to smallest (chromosome 22), and 1 pair of sex chromosomes; XX if
female and XY if male (Fig. 10). Known abnormalities that result from variations in normal
chromosome structure or number in humans are listed in Table 5.
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Figure 10. Normal human karyotype
Abnormality/disorder
(alternative name)
Symptoms Karyotype
Down syndrome
(Trisomy 21)
Cognitive disabilities, characteristic
physical features, congenital heart disease
3 copies of chromosome 21
Turner syndrome
(Gonadal dysgenesis)
Gonadal dysfunction, characteristic
physical features, congenital heart disease
1 copy of the X
chromosome
Cri du chat
(cry of the cat)
Some abnormalities include problems
with the larynx and nervous system,
resulting in a characteristic infant cry that
sounds like a meowing kitten
Truncated chromosome 5
Edwards syndrome
(Trisomy 18)
Mortality rate – 50% die within the first 2
months of life. Three times more common
in boys than girls. Birth defects include
several organ abnormalities, including
heart and kidneys
3 copies of chromosome 18
Patau syndrome
(Trisomy 13)
Mortality rate- 80%
Birth defects, including severe
neurological problem and heart defects
3 copies of chromosome 13
Table 5: Chromosomal abnormalities that can be detected by karyotype analysis
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Procedure:
A fellow scientist of BCBB Cytogeneics was assigned the task of performing karyotype
analysis for 2 infants, but he needs a second opinion before informing the parents. The karyotype
for each infant is presented below. It is your task to examine both karyotypes (#1 and #2), record
your findings in the tables provided, and then report them to your colleague.
Karyotype #1:
http://www.ratsteachgenetics.com/Genetics_quizzes/Lecture%207/7q4.jpg
CH # Remarks CH# Remarks CH# Remarks CH# Remarks
1 7 13 19
2 8 14 20
3 9 15 21
4 10 16 22
5 11 17 23
6 12 18 24
CH = Chromosome
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Karyotype #2:
https://ccr.coriell.org/images/karyotype/gm18241-xyy.jpg
CH # Remarks CH# Remarks CH# Remarks CH# Remarks
1 7 13 19
2 8 14 20
3 9 15 21
4 10 16 22
5 11 17 23
6 12 18 24
CH = Chromosome
Question:
Based on the karyotypes provided, do these babies have detectable problems in their
chromosomes? If yes, use that information to diagnose what disease/genetic abnormality
the child has.
Infant Number One Diagnosis: ________________
Infant Number Two Diagnosis: ________________
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REFERENCES:
Ben-Chetrit, E and Levy, M. (1998). Colchicine: 1998 Update. Seminars in Arthritis and
Rheumatism 28: 48-59.
Jordan, MA and Wilson, L. (2004). Microtubules as a target for anticancer drugs. Nature
Reviews 4: 253- 265.
______________________________________________________________________________
LOOK AHEAD:
• Before coming to lab next week, make sure to read the Mendelian Genetics task sheet as
well as Chapter 17 in your lab manual.
