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?

3. 2. Is DNA fully replicated?

Is DNA damage repaired?

3. Have spindle fibers formed?

Have they attached to
chromosomes correctly?
2.
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 cancer-
causing 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 single-
stranded 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 Malignant tumor (carcinoma

epithelial cells) is confined in epithelial cells) grows
by basal lamina; then very fast, becomes invasive,
additional mutation occurs. 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.