During M phase an abrupt change in the biochemical state of the cell

occurs at the transition from metaphase to anaphase; a cell can pause in

metaphase before this transition point, but once the point is passed, the
cell will carry on smoothly to the end of mitosis and through cytokinesis
into interphase.
The Cell Cycle
During development from stem to fully differentiated, cells in the body alternately
divide (mitosis) and "appear" to be resting (interphase). This sequence of activities
exhibited by cells is called the cell cycle.
Interphase, which appears to the eye to be a resting stage between cell divisions, is
actually a period of diverse activities. Those interphase activities are indispensible
in making the next mitosis possible.
Interphase: Interphase generally lasts at least 12 to 24 hours in mammalian tissue.
During this period, the cell is constantly synthesizing RNA, producing protein and
growing in size. By studying molecular events in cells, scientists have determined
that interphase can be divided into 4 steps: Gap 0 (G0), Gap 1 (G1), S (synthesis)
phase, Gap 2 (G2). Gap 0 (G0): There are times when a cell will leave the cycle and
quit dividing. This may be a temporary resting period or more permanent. An
example of the latter is a cell that has reached an end stage of development and
will no longer divide (e.g. neuron).
Gap 1 (G1): Cells increase in size in Gap 1, produce RNA and synthesize protein.
An important cell cycle control mechanism activated during this period (G1
Checkpoint) ensures that everything is ready for DNA synthesis. (Click on the
Checkpoints animation, above.)
S Phase: To produce two similar daughter cells, the complete DNA
instructions in the cell must be duplicated. DNA replication occurs
during this S (synthesis) phase.

Gap 2 (G2): During the gap between DNA synthesis and mitosis, the
cell will continue to grow and produce new proteins. At the end of
this gap is another control checkpoint (G2 Checkpoint) to determine
if the cell can now proceed to enter M (mitosis) and divide.

Mitosis or M Phase: Cell growth and protein production stop at this
stage in the cell cycle. All of the cell's energy is focused on the
complex and orderly division into two similar daughter cells. Mitosis
is much shorter than interphase, lasting perhaps only one to two
hours. As in both G1 and G2, there is a Checkpoint in the middle of
mitosis (Metaphase Checkpoint) that ensures the cell is ready to
complete cell division. Actual stages of mitosis can be viewed at
Animal Cell Mitosis.

Regulation of the Cell Cycle
The length of the cell cycle
varies depending upon the
type of cell and is extremely
important in many aspects of
biology, especially
development..
The timing differences are
primarily in the G1 phase,
which may become very long
(as in G0 phase).
Variation in the length of the
cell cycle depends upon cell
cycle checkpoints which
control the cells progression.

Restriction point
These controls make certain that the cells machinery is operating
properly with the correct timing. In addition, the cell cycle control
mechanisms must make certain that each phase of the cycle is completed
properly such that the next steps are prepared for. The control system
must be able to respond to certain conditions that may affect the cell
cycle.
The cell cycle checkpoints determine if a cell is ready to progress to the
next stage. Late in the G1 phase, the G1 checkpoint determines if the cell
will enter the following S phase. In animals, the G1 checkpoint or
restriction point, is largely controlled by growth factors.
The G2 checkpoint determines if the cell will enter the M phase and
requires the proper completion of DNA synthesis.
The third cell cycle checkpoint is the spindle assembly checkpoint which
occurs between metaphase and anaphase and requires the proper
attachment of all the chromosomes to the spindle apparatus.
The essential
processes, such as
DNA replication and
mitosis and
cytokinesis, are
triggered by a central
cell-cycle control
system. The control
system is drawn as an
indicator that rotates
clockwise, triggering
essential processes
when it reaches
specific points on the
outer dial.
The cell-cycle control system is based on two key families of proteins. The first is
the family of cyclin-dependent protein kinases (Cdk for short), which induce
downstream processes by phosphorylating selected proteins on serines and
threonines.
The second is a family of specialized activating proteins, called cyclins, that bind to
Cdk molecules and control their ability to phosphorylate appropriate target proteins
There are two main classes of cyclins: mitotic cyclins, which bind to Cdk molecules
during G2 and are required for entry into mitosis, and G1 cyclins, which bind to Cdk
molecules during G1 and are required for entry into S phase
The events that drive the cell into mitosis are as follows: Mitotic cyclin accumulates
gradually during G2 and binds to Cdk to form a complex known as M-phase-promoting
factor (MPF). This complex is at first inactive, but through the action of other enzymes
that phosphorylate and dephosphorylate it, it is converted to an active form. The ultimate
activation of MPF is almost explosive. This is believed to be due to a positive feedback
mechanism whereby active MPF increases the activity of the enzymes that activate MPF:
thus the concentration of active MPF builds up at an accelerating pace until a critical
flashpoint is reached, whereupon a flood of active MPF triggers the downstream events
that propel the cell into mitosis. MPF is inactivated equally suddenly by the degradation
of mitotic cyclin at the metaphase-anaphase boundary, enabling the cell to exit from
mitosis..
The two key subunits of MPF.
Principle CDKs and cyclins active at each stage of mammalian cycle

G1 CDK: CDK2,3,4,6 Cyclins D1-3
CDK:CDK2 Cyclins: E class [ two CDK-cyclin systems are active in
G1 of the
cell cycle.CDK2-cyclinE complex required for G1-S transiion
The other CDKs and D cyclins are responsible for
interpreting growth signals for the environment And act at the restriction
point to
channel the cell into either late G1 or G0 phase.]

S CDK:CDK2
Cyclins : A class required for DNA replication

G2/M phase CDK:CDK1
Cyclins : A and B class
The mammalian CDK1-cyclin B complex is MPF
M phase cyclins are required for Mitosis


Cyclins and Cdk proteins in the
standard vertebrate cell cycle.
Vertebrates have many different
cyclin genes and many different cdk
genes. Their products act in
different cyclin/Cdk combinations
at different stages of the cycle. The
diagram shows only a few of these
molecules and is speculative. The
roles of Cdk2 and cyclin A, in
particular, are still uncertain
Summary of feedback, size, and damage controls in the
cell cycle. The red T bars
represent checks on progress of the cell-cycle control
system arising from intracellular processes
that are uncompleted or deranged.
MUTATIONS
Changes in DNA that affect genetic
information

Gene Mutations
Point Mutations changes in
one or a few nucleotides
Substitution
THE FAT CAT ATE THE RAT
THE FAT HAT ATE THE RAT
Insertion
THE FAT CAT ATE THE RAT
THE FAT CAT XLW ATE THE
RAT
Deletion
THE FAT CAT ATE THE RAT
THE FAT ATE THE RAT



What is a mutation?
Mutation a change in the DNA of an organism
Germ-line mutation occur in gametes of organism
Passed on to offspring, do not affect the organism
Somatic mutation mutations in the organisms
body
Affect the organism, but not passed on to offspring
Ex. Skin cancer, leukemia, any cancer
Gene Mutations
Frameshift Mutations shifts
the reading frame of the genetic
message so that the protein may
not be able to perform its
function.
Insertion
THE FAT CAT ATE THE RAT
THE FAT HCA TAT ETH ERA T

Deletion
THE FAT CAT ATE THE RAT
TEF ATC ATA TET GER AT


H
H
Chromosome Mutations
Changes in number and structure of entire
chromosomes
Original Chromosome ABC * DEF
Deletion AC * DEF
Duplication ABBC * DEF
Inversion AED * CBF
Translocation ABC * JKL
GHI * DEF

Chromosome Mutations
Down Syndrome
Chromosome 21 does not
separate correctly.
They have 47
chromosomes in stead of
46.
Children with Down
Syndrome develop slower,
may have heart and
stomach illnesses and vary
greatly in their degree of
inteligence.
Chromosome Mutations
Cri-du-chat
Deletion of material on 5
th

chromosome
Characterized by the cat-like
cry made by cri-du-chat babies
Varied levels of metal
handicaps
Sex Chromosome Abnormalities
Klinefelters Syndrome
XXY, XXYY, XXXY
Male
Sterility
Small testicles
Breast enlargement
Sex Chromosome Abnormalities
XYY Syndrome
Normal male traits
Often tall and thin
Associated with antisocial and behavioral problems
Sex Chromosome Mutations
Turners Syndrome
X
Female
sex organs don't
mature at adolescence
sterility
short stature
Significance of Mutations
Most are neutral
Eye color
Birth marks
Some are harmful
Sickle Cell Anemia
Down Syndrome
Some are beneficial
Sickle Cell Anemia to Malaria
Immunity to HIV

What causes mutations?
Mutagens agent that causes
mutations to occur within a cell.
Ex. Ionizing radiation, Base analogs,
Intercalating agents, and Bromine
Types of mutations
Chromosome mutations changes in the structure
of a chromosome or loss of an entire chromosome.
Deletion loss of piece of DNA due to chromosomal
breakage
Duplication Chromosomes steal part of homologs and
have both alleles for each gene involved
Inversion piece of DNA breaks off and reattaches itself
in opposite direction
Translocation chromosome breaks off and reattaches to
a non-homologous chromosome
Nondisjunction chromosome does not properly
separate from its homolog during meiosis
Chromosome Mutations
Nondisjunction
Results in gametes receiving to many or too few
chromosomes
Ex. Down Syndrome = trisomy of chromosome 21

What kind of chromosomal mutation is this?
Original chromosome
A. Duplication
B. Translocation
C. Inversion
D. Deletion

Gene Mutations
May involve a large section of DNA or a
single nucleotide within a codon
Point mutation the substitution or change
of a single nucleotide
Insertion or Deletion one nucleotide is
removed from or added to a sequence
Frame shift mutation occurs when codons
are incorrectly grouped
DNA sequence

mRNA sequence

Polypeptide
Gene mutations which affect only
one gene
Transcription
Translation
2010 Paul Billiet ODWS

DNA (antisense strand)

mRNA


Polypeptide




Normal gene
GGTCTCCTCACGCCA

CCAGAGGAGUGCGGU
Codons

Pro-Glu-Glu-Cys-Gly
Amino acids
The antisense strand is the DNA strand which acts as
the template for mRNA transcription
2010 Paul Billiet ODWS
Point Mutation
Can result in
No effect - the
protein structure is
not changed
Missense one amino
acid is replaced by
another
Nonsense
prematurely stop
codon in amino acid
sequence
Point mutation
Ex. Sickle cell anemia mutation in a single
nucleotide that causes the malformation of the
hemoglobin molecule which carries oxygen to our
cells
Mutations: Substitutions
Substitution mutation
GGTCACCTCACGCCA

CCAGUGGAGUGCGGU


Pro-Arg-Glu-Cys-Gly
Substitutions will only affect a single codon
Their effects may not be serious unless they affect an amino acid that is
essential for the structure and function of the finished protein molecule (e.g.
sickle cell anaemia)
Normal gene
GGTCTCCTCACGCCA

CCAGAGGAGUGCGGU
Codons

Pro-Glu-Glu-Cys-Gly
Amino acids
2010 Paul Billiet ODWS
The genetic code is degenerate
A mutation to have no effect on the phenotype
Changes in the third base of a codon often have no
effect.
2010 Paul Billiet ODWS
No change
Normal gene
GGTCTCCTCACGCCA

CCAGAGGAGUGCGGU
Codons

Pro-Glu-Glu-Cys-Gly
Amino acids
Substitution mutation
GGTCTTCTCACGCCA

CCAGAAGAGUGCGGU


Pro-Glu-Glu-Cys-Gly
2010 Paul Billiet ODWS
Disaster
Normal gene
GGTCTCCTCACGCCA

CCAGAGGAGUGCGGU
Codons

Pro-Glu-Glu-Cys-Gly
Amino acids
Substitution mutation
GGTCTCCTCACTCCA

CCAGAAGAGUGAGGU


Pro-Glu-Glu-STOP
2010 Paul Billiet ODWS
Mutations: Inversion
Normal gene
GGTCTCCTCACGCCA

CCAGAGGAGUGCGGU
Codons

Pro-Glu-Glu-Cys-Gly
Amino acids
Inversion mutation
GGTCCTCTCACGCCA

CCAGGAGAGUGCGGU


Pro-Gly-Glu-Cys-Gly
Inversion mutations, also, only affect a small part of the
gene
2010 Paul Billiet ODWS
Mutations: Additions
Normal gene
GGTCTCCTCACGCCA

CCAGAGGAGUGCGGU
Codons

Pro-Glu-Glu-Cys-Gly
Amino acids
Addition mutation
GGTGCTCCTCACGCCA

CCACGAGGAGUGCGGU


Pro-Arg-Gly-Val-Arg

A frame shift mutation
2010 Paul Billiet ODWS
Mutations: Deletions
Normal gene
GGTCTCCTCACGCCA

CCAGAGGAGUGCGGU
Codons

Pro-Glu-Glu-Cys-Gly
Amino acids
Deletion mutation
GGTC/CCTCACGCCA

CCAGGGAGUGCGGU


Pro-Gly-Ser-Ala-Val

A frame shift mutation
2010 Paul Billiet ODWS
Insertion
and
Deletion
The removal or
addition of a
nucleotide base
to a sequence
usually results in
a frameshift
mutation.
Mutations of haemoglobin
Haemoglobin is a tetramer = 2 and 2 -chains
The genes for these polypeptides are found on
different chromosomes
The -chain gene is found on chromosome 11
The -chain gene is found on chromosome 16
The nucleotide sequences have been worked out
Several inherited diseases occur on the -chain,
which contains 146 amino acids.
2010 Paul Billiet ODWS
haemoglobin sense strand cDNA
sequence
cDNA (complementary DNA) is obtained
by back-transcribing the mRNA used to
translate the polypeptide
So cDNA has no introns
This is done using reverse transcriptase
enzyme.
2010 Paul Billiet ODWS
ATG GTG CAT CTG ACT CCT GAG GAG AAG TCT GCC
GTT ACT GCC CTG TGG GGC AAG GTG AAC GTG GAT
GAA GTT GGT GGT GAG GCC CTG GGC AGG CTG CTG
GTG GTC TAC CCT TGG ACC CAG AGG TTC TTT GAG TCC
TTT GGG GAT CTG TCC ACT CCT GAT GCT GTT ATG GGC
AAC CCT AAG GTG AAG GCT CAT GGC AAG AAA GTG
CTC GGT GCC TTT AGT GAT GGC CTG GCT CAC CTG GAC
AAC CTC AAG GGC ACC TTT GCC ACA CTG AGT GAG
CTG CAC TGT GAC AAG CTG CAC GTG GAT CCT GAG
AAC TTC AGG CTC CTG GGC AAC GTG CTG GTC TGT
GTG CTG GCC CAT CAC TTT GGC AAA GAA TTC ACC
CCA CCA GTG CAG GCT GCC TAT CAG AAA GTG GTG
GCT GGT GTG GCT AAT GCC CTG GCC CAC AAG TAT
CAC TAA
Methionine initiator
Nonsense terminator
2010 Paul Billiet ODWS
Mutation Codon Change to DNA
sense strand
Change in
Amino Acid
S (sickle cell
anaemia)
6 GAG to GTG Glu to Val
C (cooleys
syndrome)
6 GAG to AAG Glu to Lys
G
San

Jose
7 GAG to GGG Glu to Gly
E 26 GAG to AAG Glu to Lys
M
Saskatoon
63 CAT to TAT His to Tyr
M
Milwauki
67 GTG to GAG Val to Glu
O
Arabia
121 GAA to GTA Glu to Val
2010 Paul Billiet ODWS
Sickle Cell Anaemia
Blood smear (normal)
Image Credit: http://lifesci.rutgers.edu/~babiarz/
Sickle cell anemia
Image Credit: http://explore.ecb.org/
What affect does this mutation have
on the function of the protein?
Proteins are folded in a specific fashion
according to the amino acid sequence it
contains.
This would cause the function of the protein
to be severely reduced or not functional at
all.
Hemophilia
A sex-linked, recessive genetic disorder
that affects the individuals ability to clot
blood.
Mutations
Any change in the DNA sequence of an organism is a
mutation.
Mutations are the source of the altered versions of genes
that provide the raw material for evolution.
Most mutations have no effect on the organism, especially
among the eukaryotes, because a large portion of the DNA
is not in genes and thus does not affect the organisms
phenotype.
Of the mutations that do affect the phenotype, the most
common effect of mutations is lethality, because most
genes are necessary for life.
Only a small percentage of mutations causes a visible but
non-lethal change in the phenotype.
Mutations are Random
A central tenet of biology is that the flow of information from DNA to protein is
one way. DNA cannot be altered in a directed way by changing the environment.
Only random DNA changes occur.
The fluctuation test, an early experiment in bacterial genetics (Luria and
Delbruck, 1943) showed that variations in bacterial phenotypes are due to pre-
existing mutations and not due to physiological changes induced by environmental
conditions.
A large batch of E. coli is infected with phage T1. Most are lysed by the phage, but
a few survive. Are the survivors a few rare individuals who managed to induce
their T1-protection mechanisms on time, or are they pre-existing mutants?
--if protection is a physiological condition induced by the presence of the phage, then the
percentage of survivors will be the same among all small batches of the cells; all cells are
genetically identical.
if protection is due to pre-exisiting mutations, then some small batches will have many
survivors (descendants of T1-resistant mutants), while most other batches will have few
or no survivors (there were no T1-resistant mutants in the original batch).
Result: some batches had many survivors, but most batches had few or none. T1-
resistant mutants existed in the population before T1 was added.
Fluctuation Test
Types of DNA Change
The simplest mutations are base changes, where one base is
converted to another. These can be classified as either:
--transitions, where one purine is changed to another purine (A -> G,
for example), or one pyrimidine is changed to another pyrimidine (T ->
C, for example).
transversions, where a purine is substituted for a pyrimidine, or a
pyrimidine is substituted for a purine. For example, A -> C.

Another simple type of mutation is the gain or loss f one or a
few bases.
Larger mutations include insertion of whole new sequences,
often due to movements of transposable elements in the DNA
or to chromosome changes such as inversions or
translocations.
Deletions of large segments of DNA also occurs.
Types of Mutation
Mutations can be classified according to their effects on the protein (or
mRNA) produced by the gene that is mutated.
1. silent mutations (synonymous mutations). Since the genetic code is
degenerate, several codons produce the same amino acid. Especially, third
base changes often have no effect on the amino acid sequence of the
protein. These mutations affect the DNA but not the protein. Therefore
they have no effect on the organisms phenotype.
2. missense mutations. Missense mutations substitute one amino acid for
another. Some missense mutations have very large effects, while others
have minimal or no effect. It depends on where the mutation occurs in the
proteins structure, and how big a change in the type of amino acid it is.
Example: Hb
S
, sickle cell hemoglobin, is a change in the beta-globin gene,
where a GAG codon is converted to GUG. GAG codes for glutamic acid,
which is a hydrophilic amino acid that carries a -1 charge, and GUG codes for
valine, a hydrophobic amino acid. This amino acid is on the surface of the
globin molecule, exposed to water. Under low oxygen conditions, valines
affinity for hydrophobic environments causes the hemoglobin to crystallize out
of solution.

More Types of Mutation
3. Nonsense mutations convert an amino acid into a stop codon. The effect
is to shorten the resulting protein. Sometimes this has only a little effect, as
the ends of proteins are often relatively unimportant to function. However,
often nonsense mutations result in completely non-functional proteins.
an example: Hb- McKees Rock. Normal beta-globin is 146 amino acids long.
In this mutation, codon 145 UAU (codes for tyrosine) is mutated to UAA
(stop). The final protein is thus 143 amino acids long. The clinical effect is to
cause overproduction of red blood cells, resulting in thick blood subject to
abnormal clotting and bleeding.
4. Sense mutations are the opposite of nonsense mutations. Here, a stop
codon is converted into an amino acid codon. Since DNA outside of
protein-coding regions contains an average of 3 stop codons per 64, the
translation process usually stops after producing a slightly longer protein.
Example: Hb- Constant Spring. alpha-globin is normally 141 amino acids
long. In this mutation, the stop codon UAA is converted to CAA (glutamine).
The resulting protein gains 31 additional amino acids before it reaches the next
stop codon. This results in thalassemia, a severe form of anemia.
Frameshifts and Reversions
Translation occurs codon by codon, examining nucleotides in groups of 3. If a
nucleotide or two is added or removed, the groupings of the codons is altered. This
is a frameshift mutation, where the reading frame of the ribosome is altered.
Frameshift mutations result in all amino acids downstream from the mutation site
being completely different from wild type. These proteins are generally non-
functional.
example Hb- Wayne. The final codons of the alpha globin chain are usually AAA UAC
CGU UAA, which code for lysine-tyrosine-arginine-stop. In the mutant, one of the As
in the first codon is deleted, resulting in altered codons: AAU ACC GUU AAG, for
asparagine-threonine-valine-lysine. There are also 5 more new amino acids added to
this, until the next stop codon is reached.
A reversion is a second mutation that reverse the effects of an initial mutation,
bringing the phenotype back to wild type (or almost).
Frameshift mutations sometimes have second site reversions, where a second
frameshift downstream from the first frameshift reverses the effect.
Example: consider Hb Wayne above. If another mutation occurred that added a G
between the 2 Cs in the second codon, the resulting codons would be: AAU ACG CGU
UAA, or asparagine-threonine-arginine-stop. Note that the last 2 codons are back to the
original. Two amino acids are still altered, but the main mutational effect has been
reverted to wild type.
mRNA Problems
Although many mutations affect the protein sequence directly, it is possible
to affect the protein without altering the codons.
Splicing mutations. Intron removal requires several specific sequences.
Most importantly, introns are expected to start with GT and end in AG.
Several beta globin mutations alter one of these bases. The result is that
one of the 2 introns is not spliced out of the mRNA. The polypeptide
translated from these mRNAs is very different from normal globin,
resulting in severe anemia.

Polyadenylation site mutations. The primary RNA transcript of a gene is
cleaved at the poly-A addition site, and 100-200 As are added to the 3 end
of the RNA. If this site is altered, an abnormally long and unstable mRNA
results. Several beta globin mutations alter this site: one example is
AATAAA -> AACAAA. Moderate anemia was the result.
Trinucleotide Repeats
A fairly new type of mutation has been described, in which a particular codon is
repeated.
During replication, DNA polymerase can stutter when it replicates several
tandem copies of a short sequence. For example, CAGCAGCAGCAG, 4 copies of
CAG, will occasionally be converted to 3 copies or 5 copies by DNA polymerase
stuttering.
Outside of genes, this effect produces useful genetic markers called SSR (simple
sequence repeats).
Within a gene, this effect can cause certain amino acids to be repeated many times
within the protein. In some cases this causes disease
For example, Huntingtons disease is a neurological disease that generally strikes in
middle age, producing paranoia, uncontrolled limb movements, psychosis, and
death. Woody Guthrie, a folk singer from the 1930s, had this disease.
The Huntingtons disease gene normally has between 11 and 33 copies of CAG
(codon for glutamine) in a row. The number occasionally changes. People with
HD have 37 or more copies, up to 200). The rate of copy number change is much
higher in HD people--too many copies makes the repeated sequence more subject to
DNA polymerase stuttering during meiosis.
Interestingly, the age of onset of the disease is related to the number of CAG
repeats present: the more repeats, the earlier the onset.
Germinal vs. Somatic Mutations
Back up to the phenotype level
Mutations can occur in any cell. They only affect future
generations if they occur in the cells that produce the
gametes: these are germinal or germ line mutations.
Mutations in other cells are rarely noticed, except in the
case of cancer, where the mutated cell proliferates
uncontrollably. Mutations in cells other than germ line
cells are somatic mutations.
A human body contains 10
13
- 10
14
cells approximately.
The average mutation rate for any given nucleotide is
about 1 in 10
9
. That is, on the average 1 cell in 10
9
has that
particular nucleotide altered. This means that virtually
every possible base change mutation occurs repeatedly in
our body cells.
Retinoblastoma
Retinoblastoma is a hereditary form of cancer that
illustrates the interaction between somatic and germinal
mutations.
This disease affects the retinoblasts, cells that are
precursors to the retinal cells. Retinoblasts exist in the
eyes until about 3 years of age. Thus, retinoblastoma
always occurs by about this age.
There are 2 forms of retinoblastoma: the hereditary form,
which is almost always bilateral (affects both eyes), and
the spontaneous form, which almost always affects just
one eye. Neither parent has the disease in spontaneous
cases.
Why should the hereditary form affect both eyes while the
spontaneous form affects only 1 eye?
RB Explanation
The retinoblastoma gene, Rb, is a tumor suppressor gene. Individual
cells become cancerous if they lack this gene, or if both copies are
defective mutants: Rb
-
Rb
-
.
The mutation rate for the Rb gene is about 10
-6
, which means that
about 1 copy in 10
6
will spontaneously go from Rb
+
to Rb
-
.
The retina contains about 10
8
retinoblasts.

So, since we are diploid, a cell must go from Rb
+
Rb
+
to Rb
+
Rb
-
and
then to Rb
-
Rb
-
to become cancerous. This requires 2 independent
mutations. Since the chance of 2 independent events is the product of
the individual chances, the chance of a Rb
+
Rb
+
cell becoming Rb
-

Rb
-
is 10
-6
* 10
-6
= 10
-12
. Given that there are 10
8
retinoblasts per
person, this would occur about 1 time in 10,000 people. This is about
the rate of occurrence of spontaneous RB. That is, about 1 person in
10,000 will have 1 cell that is homozygous mutant, resulting in RB in
one eye only.
More RB Explanation
People with hereditary RB inherit one mutant
allele. Every cell in their bodies starts out Rb
+
Rb
-
. It takes only a single somatic mutation to
convert a cell to Rb
-
Rb
-
.
Given that the mutation rate is about 10
-6
and the
number of cells per retina is about 10
8
, it is almost
a certainty that multiple tumors will start in both
eyes of people with hereditary retinblastoma.
Determining the Human Mutation
Rate
Not easy. Need to look at dominant or co-dominant mutations,
because humans cant be test-crossed.
One study in Michigan looked for dominant mutations known to be
caused by a single gene, with no known phenocopies that are fully
expressed and highly penetrant. They looked at achondroplasia
(dwarfism) and retinoblastoma.
Achondroplasia: found 7 new (non-hereditary) cases among 242,257
births, for a rate of 1.4 x 10
-5
new mutations per allele per generation.
Retinoblastoma: found 52 new cases among 1,054,985 births, for a rate
of 2.3 x 10
-5
new mutations per allele per generation.
Protein electrophoresis: found 4 new alleles among 1,226,099
examined, for a rate of 3.3 x 10
-6
new mutations per allele per
generation. A bit lower than for the dominant mutations, but these
genes are smaller.
More on Mutation Rate
In 1945, at the end of World War 2, the US detonated 2 nuclear
weapons over Hiroshima and Nagasaki Japan.
Extensive studies were done of the genetic effects on the survivors. A
number of genes were examined by looking at the proteins they
produced by gel electrophoresis.
Radiation levels: a dose of 1 Sievert (Sv) is equal to 100 rem in the old
terminology.
A dose of radiation that would kill 50% of people within 60days is about 5
Sv.
Natural exposure in Chicago are is about 1 milliSievert (1 mSv) per year.
Natural exposure in Denver (5000 foot altitude) is about 1.8 mSv/year.
radiation workers are permitted up to 20 mSv per year.
average Hiroshima survivor: 200 mSv

Hiroshima Study
In the largest biochemical genetics study, 3 new
mutations affecting migration rates of proteins on
electrophoresis gels were found among 667,404
alleles examined among Hiroshima survivors.
Also, 3 new alleles were found among 466,881
alleles examined in the control group.
No easily detected change in mutation rate.
Possible to estimate radiation dose needed to
double human mutation rate at 4 Sv (with lethal
dose of 5 Sv).
However, cancer of all types is increased among
survivors and continues high to the present.
Detecting Mutagens
Radiation and certain chemical compounds are
mutagens: they cause mutation.
Cancer is caused by somatic mutations, and so
mutagens are also carcinogens.
Testing for mutagenicity is a key step is
development of pharmaceutical drugs.
Simple test using bacteria (Salmonella, a close
relative of E. coli) developed by Bruce Ames: the
Ames test.
Ames Test
Start with Salmonella that are his-, auxotrophs
unable to make their own histidine. They will
only grow if histidine is added to the growth
medium.
Add compound to be tested to growth medium,
count number of colonies growing. These are
revertants, which have been mutated back to wild
type.
In many cases, mutagens need to be activated,
converted to mutagenic state, by enzymes in the
liver that are meant to detoxify dangerous
compounds. Liver extracts are often added to the
growth medium to accomplish this.
Test isnt perfect: Salmonella are prokaryotes, and
we have complex biochemistries that modify
foreign compounds. But, it is a good initial
screen.