Announcements
1. Lab this week:
a. Bring your results from Lab 9/10 - human PCR lab. Were
you homozygous +/- Alu repeat or heterozygous? We
will use our class data for Hardy-Weinberg problem
solving this week.
b. Problem set 8, questions 1,2 due start of lab.
c. Review/problem solving; quest. 3-6 problem set 8, end of
chapter problems; lab report questions, etc…
2. Reminder to check your t-test data with Jason or myself.
Be sure you determine your unknowns correctly.
Review of lecture 37
I. Tumor suppressors - normally arrest cell cycle
II. Proto-/Oncogenes - normally promote cell cycle
III. Translocations and Genomic instability
IV. Colon cancer results from series of mutations
Learning Check
Recall that Rb (retinoblastoma) protein must be modified/
phosphorylated (+P) in order for the cell cycle to proceed.
Modification of Rb results in release of E2F. At the end of
mitosis, Rb is de-phosphorylated (-P) and binds E2F again.
1. What 2 types of proteins work together to modify/+P Rb?
2. What phenotype would you expect when cells have a
mutation such that Rb is severely truncated and can not
bind E2F?
1. Cyclins and kinases (cdks)
2. E2F will be “free” continuously to bind and activate genes
for transition to S phase - uncontrolled cell division results.
Overview of lecture 38
I. Cancer overview
- Viruses and Carcinogens
- Genetic testing for cancer
II. Population genetics
- Calculating allele frequencies
- Hardy-Weinberg law
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.
Example of virus in humans causing cancer
If infected with HBV, risk of liver cancer is increased 100x
HBV - hepatitis B virus
Normal liver cell
Infected cell - HBV genome
integrates into human
genome
1. Oncogenes activated
2. Chromosome instability
3. Cyclin genes disrupted
Tumor cell - hepatocellular
carcinoma
Carcinogens
•Chemicals are responsible for more cancers than viruses.
•Most are pro-carcinogens - must be converted metabolically to become
active carcinogens; then they bind DNA and cause point mutations
•Historically, first seen in 1700’s - scrotal skin cancer in people who
worked as chimney sweeps as children.
•Now, radiologists and farmers develop skin cancer; insulation workers
develop lung cancer, etc..
•Chemical carcinogens (tobacco smoke and diet) responsible for 50-60%
of cancer-related deaths.
-30% of cancer deaths related to smoking (cigarettes)
- the polycyclic hydrocarbons in cigarette smoke are converted
within cells and cause mutations to DNA
Genetic testing and
predicting/treating cancer
Predictive testing
Do you want to know if you have a mutation in a tumor-suppressor
gene or proto-oncogene? Could mean an increased chance of
developing cancer, but no clear answer if you will or will not get
cancer.
- what if it involves predisposition to a cancer where medical
surveillance could detect cancer early?
breast cancer vs. pancreatic cancer???
Testing for treatment/prognosis
Is there a difference in how you view a small breast tumor
depending on whether it has a mutation in p53 or not???
Know limitations and utilities of these tests
Learning Check
1) In chickens, rous sarcoma virus can cause sarcomas
(cancer) by converting the c-src proto-oncogene into v-src,
an oncogene. List 3 ways a proto-oncogene can be
converted into an oncogene?
Point mutation, translocation, overexpression
2) In humans, hepatitis B virus can cause liver cancer. In
this case, the virus can cause cancer by:
a. Converting proto-oncogenes into oncogenes
b. Causing chromosome instability
c. Disrupting cyclin genes
d. a and b
e. b and c
f. All of the above
II. Population genetics
• Studying changes in the frequencies of alleles in
populations, a subdiscipline within evolutionary
biology
• Linking Darwin’s theory of evolution to Mendel’s
genes: key insight = changes in relative abundance
of phenotypic traits can be tied to changes in the
relative abundance of alleles that influence traits
• Key to understanding genetic evolution is to focus on
populations, not individuals
Key Concepts
• Population: a group of individuals, of the same species
and location, that can actually or potentially interbreed
with each other.
• Genotype frequency: fraction of the population with a
particular genotype.
• Gene pool: all the gametes made by all the breeding
members of a population in one generation.
• Populations are dynamic: birth, death, migration,
merging populations - all lead to changes in the genetic
structure
First Step in Population Genetics Analysis:
Calculate Frequencies of Alleles
• Measurement of allele frequencies:
–Genotypes inferred directly from phenotypes
–Genotypes from DNA samples - comparing nt sequences
–Genotypes from protein samples (allozymes, next slide)
• Counting alleles from known genotypes is the easiest way.
• In simple example, there are 2 AA, 4 AB, and 2 BB
genotypes (8 total individuals, 16 total alleles):
fr(A) = (2 x 2) + (4 x 1) = 8 A/16 total = 0.5 = 50%
fr(B) = (4 x 1) + (2 x 2) = 8 B/ 16 total = 0.5 = 50%
Allozyme Analysis to Detect Genetic
Variation
• In protein gel
electrophoresis, gel is
prepared of starch,
polyacrylamide, or agarose.
• Samples move through gel
based on electric charge (no
detergent).
• Enzyme substrate added to
reveal presence of enzyme
bands.
• Allozymes are different alleles of
enzymes that can be detected
on protein gels.
• Gel showing monomorphism:
• Gel showing polymorphism:
A
B
Real-life example - calculating allele frequencies
CCR5 Function, Genotypes and Phenotypes
A small number of individuals seem to
be resistant to acquiring HIV, even
after repeated exposure. How?
Breakthrough 1996 - all have
mutations in CC-CKR-5 gene
• CC-CKR-5 gene encodes
chemokine receptor, CCR5.
• Chemokines are signaling
molecules used by the immune
system.
• HIV-1 uses CCR5 receptors to
enter host immune cells.
Allelic variation in the CCR5 gene
RFLP analysis
• 32/32 genotype associated with
resistance to HIV-1 infection.
• +/32 genotype is susceptible, but
may progress to AIDS slowly.
• +/+ genotype is susceptible to HIV-1.
32 bp deletion in exon of CCR5 gene results in
non-functional protein, and therefore resistance to HIV infection
Determine Allele Frequencies from Genotypes
How common is ∆32 allele and where is it present?
A sample of 100 French individuals in Brittany revealed the following genotypes.
Genotype:
+/+
+/32
32/32
Total
No. of individuals
79
20
1
100
1) Determining the allele frequencies by counting alleles:
No. of + alleles
158
20
0
178
No. of 32 alleles
0
20
2
22
200
Frequency of CCR5+ in sample = 178 / 200 = 0.89 = 89%
Frequency of CCR32 in sample = 22 / 200 = 0.11 = 11%
2) Determining the allele frequencies from genotype frequencies:
No. of individuals
79
20
1
100
Genotype frequency
79/100
20/100
1/100
1.00
(0.79)
(0.20)
(0.01)
Frequency of CCR5+ in sample = 0.79 + (1/2) 0.20 = 0.89 = 89%
Frequency of CCR 32 in sample = (1/2) 0.20 + 0.01 = 0.11= 11%
Conclusions and more questions
• Highest frequency of ∆32 allele is in Northern Europe; populations
without European ancestry = no ∆32
• Why is the 32 allele present in this distribution? Where did it
originate?
• Would we expect the allele to become more common where it is
presently rare?
• Use tools developed to model answers to such questions:
Godfrey H. Hardy, a mathematician, and Wilhelm Weinberg, a
physician, independently proposed a simple algebraic equation for
analyzing alleles in populations.
– Under certain conditions, one can predict what will happen to
genotype and allele frequencies
Assumptions of Hardy-Weinberg
1. No natural selection; equal rates of survival, equal reproductive
success.
2. No mutation to create new alleles.
3. No migration in or out of population.
4. Population size is infinitely large.
5. Random mating.
If these assumptions are true, then:
1. The allele frequencies in the population will not change from
generation to generation.
2. After one generation of random mating, the genotype
frequencies can be predicted from the allele frequencies.
How does such a strict law, where there is NO
change from generation to generation, help in
studying evolution?
KEY POINT: By specifying ideal conditions when
allele frequencies do NOT change, H-W law identifies
forces of evolution (forces that cause allele
frequencies to change). Know these five forces of
evolution and H-W law.
Demonstration of H-W Law
• Suppose the gene pool for a population for two
alleles is fr(A) = 0.7 and fr(a) = 0.3 in eggs and
sperm.
(Note 0.7 + 0.3 = 1)
• If random mating occurs, then what are the
probabilities that each of the following genotypes will
occur? AA, Aa, aa.
• You can solve using a Punnet square:
Calculating Genotype Frequencies
from Allele Frequencies
Sperm
fr(A) = 0.7
Eggs
fr(A) = 0.7
fr(a) = 0.3
fr(a) = 0.3
fr(AA) = 0.7 X 0.7 = fr(Aa) = 0.7 X 0.3 =
0.49
0.21
fr(Aa) = 0.7 X 0.3 = fr(aa) = 0.3 X 0.3 =
0.21
0.09
Total fr(Genotypes): 0.49 AA + 0.42 Aa + 0.09 aa = 1
What are the allele frequencies in the
next generation?
• Determine allele frequencies from genotype frequencies:
Genotype:
Frequency
AA
0.49
Aa
0.42
aa
0.09
Total
1.00
Frequency of A in sample = 0.49 + 1/2 (0.42) = 0.7
Frequency of a in sample = 1/2 (0.42) + 0.09 = 0.3
• So after one generation of random mating, the allele
frequencies can be predicted and have not changed. We’re
back where we started. No evolution of population.
General Allele and Genotype Frequencies
under H-W Assumptions
Total fr(Genotypes): p2 + 2pq + q2 = 1
Summing up H-W Equations
• Gene Pool Equation: p + q = 1 where p = frequency of the
dominant allele in the population, q = frequency of the recessive
allele in the population.
• Genotype Equation: p2 + 2pq + q2 = 1 where p2 = frequency of
dominant homozygotes, 2pq = frequency of heterozygotes, q2 =
frequency of recessive homozygotes.
KEY POINT: When population has constant allele frequencies
from generation to generation, and when genotype frequencies
can be predicted from allele frequencies, then population is in
Hardy - Weinberg equilibrium.
Three important consequences of H-W law
1. Dominant traits do NOT automatically increase in
frequency from generation to generation
2. Genetic variation can be maintained
3. Knowing the frequency of one genotype can allow for
calculation of other genotypes