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Announcements
1. Exams are graded - available after class today - in lab.
- average = 70%; 105/150
- high score = 97% (only one answer wrong)
- low score =
- see me (or email) if any questions and if you want to know your
point total to date (I have 630 or 732 pts graded so far)
2. Reminder: no labs next week. Chemotaxis lab reports due 12/6. Start
soon, so lab is fresh in your mind and you’ll have time to get answers
to questions. Work together to do t-tests. Everyone will use same
data set.
3. Genetics final exam is Monday, Dec. 9th. If you qualify to reschedule
the exam, see me ASAP.
Final is 200 pts of 1000 total pts; 1/2 of final is cumulative.
In the news…..
Titanic accident - April 14, 1912
One unidentified infant - buried in “Titanic grave”
Recently identified - how?
Very small piece of tissue (tooth with piece of root) left in
grave - most of body completely decomposed
What technique used to make ID?
Review of lecture 34
1. Drosophila behavior and genetics
2. Human behavior and genetics
3. C. elegans behavior and genetics
Overview of lectures 35/36
1. C. elegans chemotaxis behavior and genetics
2. Statistical analysis of chemotaxis data - t -tests
3. Genetics of cancer - Ch. 23
-
cell cycle regulation
mutant genes confer predisposition to cancer
tumor suppressor genes
oncogenes
translocations
genomic instability
I. Genetic approach to study chemotaxis
Paper by Bargmann et al., 1993. Cell 74: 515-527.
Known at start of study:
1. in vertebrates, olfaction is used to detect presence of any volatile
organic molecule and discriminate among different molecules
2. Odorants bind to receptors in cilia of olfactory neurons and induce a
signaling cascade in the cell
Questions: 1. How specific is interaction of odorants and receptors?
2. How many receptors are expressed on a single olfactory neuron?
3. How is information about odorants trnsmitted to brain to generate
appropriate behavior?
Approach: determine whether C. elegans is attracted to volatile organic
molecules; then screen for mutants that fail to chemotax to particular
odorant; characterization of mutants will help address questions and
allow for genes involved in process to be identified
Results of Bargmann study
-tested 121 volatile organic chemicals: 50 strong attractants
(12 alcohols), 11 variable, weak attractants; 60 not attractive
(11 alcohols)
- tested using attractance assay we’re using in lab this week
-any generalizations/ rules about which alcohols are attractive
to worms? Or is it random?
-Specific size and shape are attractive: 4-6 carbons
followed by hydroxyl group most attractive; very large
numbers of carbons are repulsive
Results, continued from Bargmann study
Is response to volatile chemicals non-specific OR is there a
specific chemical recognition of particular odorants?
How to distinguish between these two models?
Use saturation assay - expose worms to uniform
concentration of chemical 1; then add point source of
chemical 2. If attraction to chemical 2 still occurs, then
conclude a specific, saturable process is required for
chemotaxis to each chemical.
- 7 classes of volatile organic chemicals that are
likely recognized by different receptor proteins
Characterization of odr mutants
Many different classes of mutants isolated:
-some affect responses to all classes of volatile chemicals
- what kind of protein affected?
-some affect a subset of responses mediated by one
neuron type
- ex. odr-4 mutant does not respond to diacetyl but
does respond to pyrazine (specificity in defect)
- what kind of protein affected?
- results from a different study on chemotaxis identified the
odr-10 mutant; it is also defective specifically to diacetyl
- what kind of protein affected?
Do these “lab behaviors” relate to
behavior in wild?
Why does C. elegans chemotax to both water-soluble and
organic, volatile chemicals?
- short range to find nearby bacteria (food)
- long range to find more distant food sources
Why does C. elegans avoid certain chemicals?
(ex. high osmolarity solutions)
- they can cause paralysis and death
II. Statistical analysis of chemotaxis data
Is there a significant difference between each index of each
unknown and WT?
III. Genetics of Cancer
Is cancer a single disease?
In all cancers, mutations that alter gene expression are seen
Most such mutations are somatic; 1% are germ-line - what is the
difference?
Cancer “runs in families” - known for over 200 years
- but no clear-cut pattern of inheritance
- usually one mutant allele of a cancer-causing gene is
inherited - predisposes person to cancer
- likelihood cancer develops depends on particular mutant
allele, mutations in other genes, and environmental factors
Mutations play a central role in cancer
-background rate of spontaneous mutation - due to ?
- therefore, always baseline rate of cancer
-above baseline, environmental agents that promote
mutation also contribute to cancer = carcinogens
• Which mutant genes are most likely to result in cancer?
• How many mutations are needed to cause cancer?
• How do mutations convert normal cells into malignant
tumors? What are the differences between these cells?
1) uncontrolled growth 2) metastasis
The “cell cycle”
Many cells alternate between dividing and “resting” or not dividing
Gap 1; metabolic activity and cell growth
G0 (resting phase)
Mitosis
DNA synthesis
1 hour of 16 hour
cell cycle
Gap 2; metabolic activity and cell growth
Three main checkpoints in the cell cycle
•2001 Nobel Prize was awarded to 3 scientists who studied
genes that regulate the cell cycle
1.
1. Is cell the correct size?
Is DNA damaged?
2. Is DNA fully replicated?
Is DNA damage repaired?
3.
2.
3. Have spindle fibers formed?
Have they attached to
chromosomes correctly?
Why are cell cycle checkpoints important?
What might result if DNA repair has not finished?
Uncontrolled cell division could occur - cancerous cell
Example: p53 protein normally targets cells with severe
DNA damage to undergo programmed cell death.
(this removes them from the population)
If the p53 gene is mutated, damaged cells will not
be removed and may continue dividing in an
uncontrolled manner.
Many different types of cancers involve mutations of p53.
Checkpoint Control of Cell Cycle
Cdk-G1 cyclin
Cdk-Mitotic cyclin
(MPF)
Retinoblastoma
• Diagnosis: “Cat’s eye”
reflection (leukocoria) in
affected eye.
• Most common cancer of
infants and children
(1/20,000 U.S. live births).
• Survival > 90% with early
diagnosis and treatment.
• Individuals at greater risk of
developing other cancers.
Retinoblastoma Gene
• A Tumor Suppressor, which normally suppresses
unregulated cell growth.
• Discovered as a regulator of growth of neuroblasts in
developing retina of the eye.
• Inactivation of both copies of the Rb Gene removes a
“brake” on growth, leading to increased incidence of
retinal cancer.
• Since found to be active in all cells.
Retinoblastoma: Familial v. Sporadic
“Loss of
Heterozygosity”
Common
Rare
Rb Protein is Inactivated By CDK-Cyclin
During G1  S
p53 Gene (tumor suppressor)
Normal Functions
• The “Last Gatekeeper” gene since malignant state
not attained despite the presence of other cancercausing mutations until p53 is inactivated by
mutation.
• Acts as a Transcription Factor to activate expression
of p21, which inhibits CDK/G1 cyclin to halt the cell
cycle.
• Activates DNA repair.
• Triggers apoptosis (programmed cell death) if
damage can’t be repaired.
Role of p53 in
Cell Cycle
Control
p53 Mutations
• Most commonly mutated gene in cancers (50% of
total).
• When p53 mutates, DNA-damaged cells are not
arrested in G1 and DNA repair does not take place.
This failure to arrest DNA-damaged cells will be
repeated in subsequent cell cycles permitting other
mutations to accumulate, culminating in neoplastic
transformation... tumor formation and cancer.
Breast Cancer Tumor Suppressors
• A small proportion of breast cancer is heritable. Two
genes are associated with predisposition to breast
cancer.
– BRCA1 on chromosome 17
– BRCA2 on chromosome 13
• Normal function of both is in repair of ds DNA breaks.
Oncogenes
• Arise from mutation in normal gene called a
proto-oncogene.
• Dominant mutation: one copy is sufficient to
cause cancer.
• First link between viruses and cancer
proposed by Francis Peyton Rous in 1910
(Nobel Prize, 1966): cell-free extracts from
chicken tumors injected into healthy chickens
caused new tumors.
Rous Sarcoma Virus (RSV)
• Discovered by Harold Varmus and Bishop, 1975-76
(Nobel Prize, 1989).
• A transforming retrovirus: a cancer-causing singlestranded RNA virus that uses reverse transcriptase
enzyme to make ssDNA, then ds DNA, which
integrates into host DNA.
• Note: not all retroviruses are TRV’s, not all oncogenes
caused by viruses.
• 100’s of oncogenes now known.
• Human T-cell leukemia virus (HTLV) is only human
TRV known; codes a TF.
Southern Blots Probed with viral src Gene
Revealed Cellular Origin of Oncogenes
Infected chicken #1 Infected chicken #2 Uninfected chicken
(Negative Control)
v-src
c-src
Proto-oncogene
SURPRISE!
Origin of Transforming Retroviruses
Capsid protein Reverse Transcriptase Envelope Protein
Mutation creates oncogene
Ras Proto-oncogene
• Mutated in 30% of all cancers.
• A “molecular switch” in the signal transduction
pathway leading from growth factors to gene
expression controlling cell proliferation: GF 
receptor   Ras    TF  target genes 
growth.
• A single amino acid change in Ras protein can cause
constant stimulation of the pathway, even in the
absence of growth factors.
Cancers Usually Result from a
Series of Mutations in a Single Cell
• Colon Cancer:
oncogene
oncogene
Tumor suppressors
Tumor Progression: Evolution at the
Cellular Level
Benign tumor (polyp in
epithelial cells) is confined
by basal lamina; then
additional mutation occurs.
Malignant tumor (carcinoma
in epithelial cells) grows
very fast, becomes invasive,
and metastasizes.
Cancer Cells Evade Two “Safety”
Mechanisms Built into the Cell Cycle
1. Once p53 is inactivated, cells with DNA damage don’t
arrest from G1 and don’t undergo apoptosis.
2. Telomerase enzyme is activated, avoiding the limit to
cell divisions imposed by telomere shortening.