Mutations

1
Mutations
mutation any heritable change in the genetic
material from any source spontaneous mutation
statistically random, unpredictable change
usually caused by errors in replication induced
mutation mutation that occurs after exposure to
radiation or various chemicals appear same
as spontaneous mutations do- origin is
different rate of mutation probability of a
mutation in a single generation varies by
organisms RNA viruses mutate very quickly
organisms without proofreading also mutate
quickly
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Mutations
somatic mutations mutations in multicellular
organisms where the change cannot be passed on
to the offspring-- results in mosaic or
chimeric individuals (usually fewer cells than X
inactivation)
germ-cell mutation mutations in cells that
ultimately produce gametes only ones that can
be passed on to future generations
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Mutations
conditional mutations mutations that only show
up under some circumstances restrictiv
e condition circumstances when a conditional
phenotype is seen permissive condition
circumstance when a conditional phenotype is
invisible temperature sensitive mutation
conditional mutant dependent on temp usually
heat sensitive (ie. phenotype seen hot), can also
be cold sensitive
BRCA1 inactive
BRCA1 active
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Mutations
temperature sensitive mutations are probably the
most common conditional mutants many were
isolated in yeast and bacteria where they would
grow at low temperature (permissive condition)
and not at higher temperatures Siamese cats also
have a temperature sensitive mutation-- black
color is produced by a pigment which is only
active in relatively cooler body parts-- tail,
paws, ears, etc.
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Mutations
mutations can also be classified by their effect
on gene function null mutation gene function is
totally absent also called loss of function
mutations, knockout mutations, deletions, hypomor
ph mutation which reduces but does not eliminate
gene function can be either a change in the
amount of protien or activity of the
enzyme hypermorph mutation which increases
levels of gene function, usually by increasing
the amount of protein expressed
ectopic new place
null mutant no pigment
hypomorph less color
hypermorph more color
gain function bluered
normal
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Mutations
gain of function mutation significant alteration
in the action of a gene can be a change in
substrate activity or expression in a new place
where the protein is not normally
found ectopic expression expression of a
wildtype gene in a new location
green fluorescent protein is usually in
jellyfish, not tadpoles-- ectopic expression
knotted1 gain of function mutation
normal leaf of arabidopsis
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Mutations
base substitution or conversion one nucleotide
is changed into another missense mutation
substitute 1 amino acid for another most
common type of protein mutation silent
mutationchange in the gene which doesn't change
the protein
AUG GTC AAT AAA CCG... met val asn lys pro
normal
AUG GTC AAG AAA CCG... met val lys lys pro
AUG GTC AAT AAC CCG... met val asn lys pro
nonsense mutation generates a new stop codon
stops protein synthesis and is often a null allele
AUG GTC AAT TAA CCG... met val asn OCR
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Mutations
frameshift mutation base pair addition or
deletion that alters the nucleotides that make
up each codon AUG GTC AAT AAA CCG... met val asn
lys pro AUG TGT CAA TAA ACC G... met cys gln
OCR AUG TTG TCA ATA AAC CG... met phe ser ile
asn AUG TTT GTC AAT AAA CCG... met phe val asn
lys pro
most frameshifts eventually result in protein
truncations
adding or deleting multiples of 3 do NOT change
the frame
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Mutations
selection process of giving a survival advantage
to certain individuals some mutations are
advantages positive selection selecting FOR
something, ie. herbicide resistance negative
selection selecting AGAINST something, ie.
another marker when used in combination, can be
a useful tool for generating new phenotypes
that are lacking the experimental marker people
generally don't want new proteins being added
to their food supplies
positive selection
genes on different chromosomes
negative selection
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Mutations
one common mutation is converting cytosine to
thymine cytosine can become methylated as a
normal cellular event full function not
entirely clear, but there is less transcription
from highly methylated DNA-- ie. X
inactivation has methylated DNA cytosine can
also undergo a deamination reaction- loss of an
amino group methylcytosine losing an amino group
thymine (ie. now a mismatch) can be
repaired by mismatch repair enzymes to either GC
or AT
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Mutations
depurination loss of a purine base from sugar
naturally labile (cleavable) in H2O 10x more
common than any other mutation
base analog chemical that looks like a DNA base
that can become incorporated into DNA (ie.
like AZT is used by reverse transcriptase)
bromodeoxyuridine base analog used to label
dividing cells- reads just like thymine but
not removed
5'-TAGCCATCTAGAATTCCGCTAGGC-3'
3'-AUCGGUAGAUCUUAAGGCGAUCCG-5'
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Mutations
acridine molecules planar molecules which cause
single base insertions or deletions causing a
frameshift mutation UV light causes mutations by
being absorbed by base pairs often causes
thymine-thymine dimers where 2 adjacent T's
become bonded on the same DNA strand T-T
dimers block both transcription as well as
replication until repaired causes a bulge in
the DNA xeroderma pigmentosum disease where
the UV damaged DNA repair system is broken
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Mutations
ionizing radiation is a powerful mutagen (X-rays,
etc) generates free radicals which can attack
DNA can cause single strand breaks, double
stranded breaks, and nucleotide
alterations cells undergoing mitosis are more
likely to suffer chromosomal breaks from
ionizing radiation-- this is why it is used in
cancer treatment after Chernobyl, mutation rates
in exposed individuals doubled in 3 of 5
studied loci animals around Chernobyl also
showed increased mutation rate could be caused
by chemical or radioactive mutagens environmental
factors can play a major, often dominant, role
in causing mutations and genetic damage
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DNA Repair Mechanisms
mismatch repair fixes base pairs that are not
forming the normal pattern of double bonds a
major role of mismatch repair is to fix incorrect
base pairs during replication-- fixes 99.9 of
errors even after proofreading corrections
preferentially 'fixes' the less methylated
strand otherwise picks randomly
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DNA Repair Mechanisms
mutS recognizes the base error and recruits
mutL mutH nicks DNA at a nearby GATC sequence
and allows a nuclease to degrade the DNA
around the error DNA polymerase uses the
remaining DNA as a primer and fills the
gap DNA ligase fixes the nick eventually DNA
gets methylated if repairing other errors,
methylation is equal and system chooses
randomly
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DNA Repair Mechanisms
deamination reaction of methylcytosine gives
thymidine deamination of normal cytosine yields
uracil DNA uracil glycosylase removes uracil
from the deoxyribose sugar leaves a guanine
unpaired with just a sugar-phosphate backbone AP
endonuclease system repairs DNA where the base
has been lost from the sugar, either through
depurination or DNA uracil glycosylase
endonuclease cleaves the backbone without a
base DNA polymerase followed by DNA ligase
repairs the gap
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DNA Repair Mechanisms
excision repair fixes errors which result in a
distortion of the double helix like thymine
dimers recognized during transcription by
RNA polymerase two nicks are made in the strand
of DNA that is defective that strand is now
free to leave DNA polymerase fills the gap 5' to
3' DNA ligase seals the last nick excision
repair is similar to mismatch repair except
1)it does not use mutS and mutL 2) usually
makes a smaller gap-- doesn't require GATC
sequence
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DNA Repair Mechanisms
postreplication repair repair of DNA damage
requiring replication DNA polymerase stalls at
certain damaged sites, like thymine dimers DNA
polymerase can skip over the damaged region
making a gap, then switch strands (with the same
polarity) and repair the gap with polymerase
followed by DNA ligase
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Mutagen Testing
Ames test looking for a reversion mutation that
converts a bacteria from a his- phenotype to a
wildtype his phenotype reversion mutation
change from a mutant phenotype back to wild
type chemicals that cause mutations will
increase the frequency of finding reversion
mutations more potent mutagens cause
reversions at lower concentrations Ames test
gives a quantitative measure of mutagenic
potency almost all carcinogens are also
mutagens in the Ames test
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Molecular Mechanisms of Recombination
mismatch repair enzymatic DNA degradation and
re-synthesis when an inappropriate base pair
combination is detected random choice as to
which is the wrong DNA-- one correct, one
mutant heteroduplex equal exchange of DNA
strands where there is a small difference
between two chromosomal strands
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Molecular Mechanisms of Recombination
Holliday model of recombination nicked DNA is
initially rejoined to the wrong chromosome,
generating a 4 stranded hybrid
nick break in the sugar phosphate backbone
of 1 strand of DNA while the other remains
intact
because the chromosomes are almost identical
paired strands can switch (strand invasion)
4 stranded intermediate can then be 'fixed' in
two ways if spliced, one DNA strand is
rejoined to a different one
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Holliday junction in an electron micrograph
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Transposable Elements
there are ears of corn where some kernels are
dark and others light the DNA responsible for
the color shift does not have a constant
location in the genome and can cause DNA
breakage transposition movement of DNA from
one position to another transposable element
piece of DNA that can move around in the
genome transposable elements are very common--
related to 50 of genome!!! not all
transposable elements are able to keep moving
not able to move all the time, many have
undergone extensive mutation
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Transposable Elements
usually flanked by direct or inverted repeats
10-200 bp in length often results in target
site duplication after integration transposase
enzyme which catalyzes transposition--
specific for each family of transposable
elements most transposable elements encode
their own transposase-- those that don't
require another transposon-- trans-activation
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Transposable Elements
Orientation of DNA repeats determines what will
happen if sequences undergo homologous
recombination direct repeats cause excision,
inverted repeats cause inversions
recombination between elements often cause
reciprocal translocations
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Transposable Elements
LTR retrotransposons family of transposable
elements distantly related to RNA
viruses contains long terminal repeats of
200-500 bases at each end move to different
sites using an RNA intermediate and reverse
transcriptase to make new DNA for
insertion unlike the other type of transposon,
these make additional copies of themselves
without moving
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Transposable Elements
many families of retrotransposons are known, and
make up a lot of the human genome
repeats and retrovirus like elements make up the
bulk of heterochromatin
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Complementation Test
start with 2 heterozygous individuals for a
phenotype of interest
if a and a are really the same gene, you see the
recessive phenotype if a and a are
different genes (ie. a and B), no recessive
phenotypes are observed because both genes will
be present from the other parent
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Complementation Test
if no recessive phenotypes are shown when
individuals heterozygous for the same phenotype
are mated, the genes complement each other
often done using bacteria or yeast-- strains that
can exist with only 1 copy of the gene and
thus always showing the phenotype gametes then
fuse, giving either 1 normal 1 mutant copy of
each gene or 2 mutant copies of 1
gene complementation tests define a functional
gene phenotypes that fail to complement each
other are in the same gene if phenotypes
do complement each other, they are different genes
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Complementation Test
given the following 7 separate strains of mutants
for histidine synthesis A B C D E F G A - -
B - - - C - - D - E
- - F - G - mutant strains
mated to themselves MUST fail to complement--
they are, by definition, the same
gene because A and D fail to complement, they
are also the same gene (AD) same goes for BCG
and EF therefore 3 separate genes are shown
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Complementation Test
you can also work logically to figure out what
genes should complement A B C D E F G A __ _
_ - - B __ __ C __ __ __ - D __
__ E __ __ __ F __ __ G __
-

-

-


-

-

-
-

-
mutants cannot be different than themselves,
therefore fail to complement
from the chart, AE, AF, and CG because they
fail to complement since DltgtF, then DltgtE because
EF also AltgtD because DltgtF
since CG and BltgtG and DltgtG, then BltgtC and CltgtD
since CltgtE and AEF, then CltgtF
since AE and AF, then EF since AEF and
AltgtG, then (E F)ltgtG