Lab 7: Cell Division: Mitosis and Meiosis

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Lab 7: Cell Division: Mitosis and Meiosis
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
Are there molecular
signals in the cytoplasm
that regulate the cell
cycle?
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
Are there molecular
signals in the cytoplasm
that regulate the cell
cycle?
Conclusion: The results suggest that
molecules are present in the cytoplasm of
cells in the S or M phase controlling the
progression of phases.
What are these molecules you ask?
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
The cell cycle clock:
Cyclins and Cyclin-Dependent
Kinases
What is a cyclin-dependent kinase
(cdk)?
It’s a group of related kinases (enzymes
that phosphorylates molecules – usually
proteins) that are activated by a group of
proteins called cyclins.
What is a cyclin?
A group of proteins that are made in
cycles (hence the name) and are
allosteric activators of the cdk’s (activate
them upon binding).
Ex. “Cyclin B”
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
Expression of human cyclins throughout the cell cycle
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
The cell cycle clock:
Cyclins and Cyclin-Dependent
Kinases
Give an example:
Ex. “Cyclin B” (the cyclin in the figure to
the right) begins to be made at the end
of S phase and slowly increases in
concentration throughout G2.
When the concentration gets high
enough, Cyclin B will bind to Cdk-1 (the
Cdk shown in the diagram to the right),
activating it.
“Cyclin B” + Cdk-1 = MPF
MPF stand for maturation-promoting
factor –(aka mitosis-promoting factor).
****All molecular binding (binding of two
molecules) is concentration dependent.
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
The cell cycle clock:
Cyclins and Cyclin-Dependent
Kinases
Give an example:
Ex. MPF will activate mitosis at the G2
checkpoint by going around and
phosphorylating a number of proteins
including:
1. Condensins
Proteins involved in condensing the chromosomes.
2. Histones – to condense DNA
3. Proteins involved in microtubule
spindle fiber formation
4. Lamins
The proteins that hold the nuclear
envelope together (nuclear lamina)
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
The cell cycle clock:
Cyclins and Cyclin-Dependent
Kinases
Give an example:
How will the MPF complex be turned off?
High levels of MPF result in the
phosphoylation of proteins that will
degrade (break down) Cyclin-B negative feedback
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
All of the various cyclins and Cdk’s are part of the…
Cell Cycle Control
Lab 7: Cell Division: Mitosis and Meiosis
Recall that mitotic cell division is regulated
by Cyclin and Cdk’s (cyclin dependent
kinases).
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
Checkpoints of the Cell Cycle
There are three
major
Control
System
checkpoints:
A. G1 checkpoint
- Passing G1 checkpoint = point of
no return
- Cell can halt at this checkpoint
and remain there for their lifetime
-external and internal signals
required to pass this checkpoint
-Growth Factor (external signal) like
PDGF (platelet derived growth
factor), which stimulates division near
a wound.
- Enough nutrients/enzymes (internal)
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
Checkpoints of the Cell Cycle
There are three
major
Control
System
checkpoints:
B. The G2 checkpoint
Ex) Is all the DNA replicated?
Ex) Is the DNA too damaged from replication
(too many mistakes made) to continue? If yes,
apoptosis; if no, continue to mitosis.
-Internal signals only
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
Checkpoints of the Cell Cycle
There are three
major
Control
System
checkpoints:
C. The M (metaphase)
checkpoint
Ex) Are all the sister chromatids
present and attached to
microtubules and ready to be
pulled apart?
-internal signals only
Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles
AIM: Describe how the cell cycle is regulated.
Checkpoints
Review
Lab 7: Cell Division: Mitosis and Meiosis
Lab 7: Cell Division: Mitosis and Meiosis
Lectin
- Highly specific carbohydrate-binding
proteins
- Present throughout nature
Ex) Concanavalin A
Jack Bean ConA – pdb code: 3CNA
Jack Bean
- Promotes cell division in plant cell by binding to cell surface receptors
that have been glycosylated with very specific polysaccharide chains.
Lab 7: Cell Division: Mitosis and Meiosis
What’s a Lectin?
Ex) Concanavalin A
Jack Bean ConA – pdb code: 3CNA
Jack Bean
Lab 7: Cell Division: Mitosis and Meiosis
Lab 7: Cell Division: Mitosis and Meiosis
Lab 7: Cell Division: Mitosis and Meiosis
After the procedure you
should see this for both the
lectin-treated (experimental)
and untreated (control)
groups:
Lab 7: Cell Division: Mitosis and Meiosis
Lectin-Treated (Expt) Untreated (Control)
Data from one group:
Root Tip
Interphase
Mitotic
Total
1
78
21
100
2
92
35
127
3
64
15
79
Total
234
71
306
Root Tip
Interphase
Mitotic
Total
1
84
32
116
2
102
44
146
3
91
39
130
Total
277
115
392
Lab 7: Cell Division: Mitosis and Meiosis
Untreated (Control)
Lab 7: Cell Division: Mitosis and Meiosis
Root Tip
Interphase
Mitotic
Total
1
78
21
100
2
92
35
127
3
64
15
79
Total
234
71
306
Lectin-Treated (Expt)
Percentage of cell in interphase234/306*100= 76.5 %
= now know to expect that in a standard onion plant,
We
76.5% of the cells will be in interphase….
Root Tip
Interphase
Mitotic
Total
1
84
32
116
2
102
44
146
3
91
39
130
Total
277
115
392
Therefore, if we have 392 total cells, we expect, if
standard,
300 cells – expected value
Lectin-Treated (Expt)
Lab 7: Cell Division: Mitosis and Meiosis
Root Tip
Interphase
Mitotic
Total
1
84
32
116
2
102
44
146
3
91
39
130
Total
277
115
392
300 cells in interphase – expected value
Therefore 92 cells in mitotic phase – expected value
Interphase
Mitotic Phase
Observed
277
115
Expected
300
92
Now calculate the chi2 and determine a p-value…
Lab 7: Cell Division: Mitosis and Meiosis
Interphase
Mitotic Phase
Observed
277
115
Expected
300
92
Now calculate the chi2 and determine a p-value…
= (277-300)2/300 + (115-92)2/92
= 529/300 + 529/92
DOF = # groups – 1 = 2 -1 = 1
= 1.76 + 5.75
P < .01
= 7.51
There is less than 1% probability that
there is no difference (null hypothesis).
Therefore, there is >99% that there is a
difference and therefore the data supports
the alternative hypothesis.
Lab 7: Cell Division: Mitosis and Meiosis
Lab 7: Cell Division: Mitosis and Meiosis
Lab 7: Cell Division: Mitosis and Meiosis
Lab 7: Cell Division: Mitosis and Meiosis
Hela cell karyotype
Normal cell karyotype
Lab 7: Cell Division: Mitosis and Meiosis
Hela cell karyotype
Lab 7: Cell Division: Mitosis and Meiosis
HPV – Human Papillomavirus
-most common STD
- Infects genital areas as well as
mouth and throat
- Nearly everyone gets it at
some point in their lives
- 90% of cases fought off by
immune systom and no
symptoms arise.
- The other 10%:
- Genital Warts
- Cervical Cancer
*Nearly all cases of cervical cancer are caused by HPV!!!
Lab 7: Cell Division: Mitosis and Meiosis
How does HPV cause cancer?
- HPV is a small DNA virus
- Ligand/receptor interactions
allow virus access to cell.
- Genome of virus enters
nucleus by unknown
mechanism
- Between 10 and 200 copies
are inserted into persons
-nuclear
ProteinDNA.
E6 and E7 are the
major oncogenic (cancer
causing)
proteins
- “E” stands
for early because
the gene is transcribed early in
the viral life cycle. “L” for late.
HPV genome - ~8000bp
Lab 7: Cell Division: Mitosis and Meiosis
Why are E6 and E7 oncogenic?
E6 functions by ubiquitinating
p53Recall that adding the small
protein ubiquitin to a protein
targets it to be degraded by the
proteosome!
Both p53 (guardian angel) and pRb are
tumor suppressor proteins.
Lab 7: Cell Division: Mitosis and Meiosis
p53
- Transcription factor
- Goes to work when the cell is in bad shape (DNA damage,
etc…)
- Like batman
- Turns on genes that code for proteins having to do with
DNA repair, cell cycle arrest and apooptosis.
p53 (“the guardian of the
genome”) bound to an enhancer
region
of DNAits apparent mass
“53”
because
is 53kDa or 53,000Da or
53,000amu.
Lab 7: Cell Division: Mitosis and Meiosis
pRb
- Inhibits cell cycle progression at G1 checkpoints
- Binds and inhibits transcription factors that want to turn on
genes and push the cell into S phase.
pRb (Retinoblastoma Protein)
- Named such because if you both copies
are mutated early in life you will get
retinoblastoma (cancer of the eye’s retina)
pRb is inactivated
when it is
phosphorylated by
CDK/cyclin!!
Lab 7: Cell Division: Mitosis and Meiosis
Lab 7: Cell Division: Mitosis and Meiosis
http://www.youtube.com/watch?v=pOROHmEmqmU
Focus on meiosis part

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