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Mutation
Mutation
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 organism’s 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.
• 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 108
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 can’t 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 isn’t perfect: Salmonella are prokaryotes,
and we have complex biochemistries that
modify foreign compounds. But, it is a good
initial screen.