Download Mutations

Genetics 2011
Outline of Chapter 7
• What mutations are
• How often mutations occur
• What events cause mutations
• How mutations affect survival and evolution
Lecture 7
Anatomy and Function of
a Gene: Dissection
Through Mutation
• Mutations and gene structure
• Experiments using mutations demonstrate a gene is a
discrete region of DNA.
• Mutations and gene function
ᯘ⩶ె ⪁ᖌ
• Genes encode p
proteins by
y directing
g assembly
y of amino
acids.
http://lms.ls.ntou.edu.tw/course/136
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• Mutations that alter g
genes’ instructions for amino
acids alter protein structure and function.
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Genetics 2011
Mutations: Primary tools of genetic
analysis
Classification of mutations by affect on
DNA molecule
• Mutations are heritable changes in base
sequences that modify the information
content of DNA.
• Forward mutation – changes wild-type to
different allele
• e.g. A+ Æ a or b+ Æ B
• Reverse mutation – causes novel mutation
to revert back to wild-type (reversion)
• e.g. a ÆA+ or B Æ b+
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• Substitution – base is replaced by one of the
other three bases
• Deletion – block of one or more DNA pairs is
lost
• Insertion – block of one or more DNA pairs is
added
• Inversion 1800 rotation of piece of DNA
• Reciprocal translocation – parts of
nonhomologous chromosomes change
places
• Chromosomal rearrangements – affect many
4
genes at one time
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Mutations classified by their effect on DNA
Genetics 2011
Spontaneous mutations influencing
phenotype occur at a very low rate.
Rates of recessive forward mutations at five coat color
genes in mice
ƒ 11 m
mutations
tations per gene e
every
er 106 gametes
Mutation rates in other organisms
ƒ 2 – 12 mutations per gene every 106 gametes
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Are mutations spontaneous or
induced?
G
General
l observations
b
ti
off mutation
t ti
rates
t
Mutation rates are <10
10-9 to >10
10-3 per gene per gamete
• Differences in gene size
• Susceptibility of particular genes to various
mutagenic mechanisms
Average mutation rate in gamete-producing
eukaryotes
y
is higher
g
than that of p
prokaryotes
y
• Many cell divisions take place between zygote
formation and meiosis in germ cells
ƒ More chance to accumulate mutations
• Can diploid organisms tolerate more mutations
than haploid organisms?
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•
•
•
•
Mutation: random events!
Biochemical response: inducible event!
M t mutations
Most
t ti
are spontaneous.
t
p
-a
Luria and Delbruck experiments
simple way to tell is mutations are
spontaneous or if they are induced by a
mutagenic agent
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The Luria
Luria--Delbrü
Delbrück
ck fluctuation experiment
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Replica plating verifies preexisting
mutations
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Interpretation of Luria-Delbruck
Luria Delbruck fluctuation
experiment and replica plating
Bacterial resistance arises from mutations that
occurred before exposure to bactericide
Genetics 2011
Chemical and Physical agents cause
mutations.
• Hydrolysis of a purine
base, A or G
occurs:1000/hr in every
cell
• Bactericide becomes a selective agent
• Kills
Kill nonresistant
i t t cells
ll
• Allows survival of cells with pre-existing
resistance
• Deamination removes –
NH2 group. Can change
C to U, inducing a
substitution
b tit ti tto and
dA
A-T
T
base pair after replication
M t ti
Mutations
occur as the
th resultlt off random
d
processes
• Once such random changes occur
occur, they usually
remain stable
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Fig 7 6 a b
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• X rays break the
DNA backbone
backbone.
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Oxydation
y
from free radicals formed by
y
irradiation damages individual bases
• Irradiation causes formation of free radicals (e.g.
reactive oxygen) that can alter individual bases
• 8-oxodG mispairs with A
• Normal G-C Æ mutant T-A after replication
• UV light produces
thymine dimers.
Fi 7.6
Fig.
7 6 c, d
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Fig. 7.6 e
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Repair enzymes fix errors created by
mutation.
Excision
E
i i repair
i
enzymes
release
damaged
regions of
DNA. Repair
is then
completed by
DNA
polymerase
and DNA
ligase
ligase.
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Mi t k d
Mistakes
during
i
DNA replication
li ti
Incorporation of incorrect bases by DNA polymerase
is exceedingly rare (<
( 10-9 in bacteria and humans)
Two ways
y that replication
p
machinery
y minimizes
mistakes
• P
Proofreading
f di function
f
i off DNA polymerase
l
(Fi
(Fig 7
7.7)
7)
ƒ 3'-to-5' exonuclease recognizes and excises
mismatches
• Methyl-directed
y
mismatch repair
p ((later in this chapter)
p )
ƒ Corrects errors in newly replicated DNA
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DNA polymerase proofreading
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Unequal crossing over can occur between
homologous chromosomes
Pairing between homologs during meiosis can be
out of register
• Mispaired base is
recognized and
excised by 3'-to-5'
exonuclease of
DNA polymerase
Unequal
q
crossing-over
g
results in a deletion on one
homolog and a duplication on the other homolog
• Improves fidelity of
replication 100-fold
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Fig. 7.7
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Colorblindness may occur from this!
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Transposable elements move around the genome and are
not susceptible to excision or mismatch repair.
• TEs can "jump" into a gene and disrupt its
function
• Two mechanisms of TE movement (transposition)
Fig. 7.8 b
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7.8 aLectured by Han-Jia Lin
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Trinucleotide instability causes
mutations.
• FMR-1 genes in
unaffected
people have
fewer than 50
CGG repeats.
• Unstable
premutation
alleles have
between 50 and
200 repeats.
• Disease
causing alleles
have > 200
CGG repeats.
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Fig. B(1) Genetics and Society
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Trinucleotide repeat in people with
fragile X syndrom
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Experimental evidence that mutagens
induce mutations
• Mutagens can be used to increase mutation
rates
• May be physical or chemical
• H. J. Muller – first discovered that X rays
y
increase mutation rate in fruitflies
• Exposed
p
male Drosophila
p
to large
g doses of X rays
y
• Mated males to females with balancer X chromosome
(dominant Bar eyed mutation and multiple inversions)
• Could assay more than 1000 genes are essential to
Drosophila viability on the X chromosome
Fi A,
Fig.
A B(2) Genetics
G ti andd Society
S i t
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Mutagens increase mutation rate using
different mechanisms.
M ll ’ experiment
Muller’s
i
t
Find out lethal mutants on X chromosome of Drosophila
Fig. 7.9
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Replace
R
l
ab
base: B
Base analogs
l
- chemical
h i l structure
t t
almost identical to normal base
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Fig. 7.10
a
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by Han-Jia
Lin
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How mutagens alter DNA:
Chemical action of mutagen
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How mutagens alter DNA:
Chemical action of mutagen (cont)
Alter base structure and properties:
H d
Hydroxylating
l ti agents
t add
dd an –OH
OH group
Alter base structure and properties (cont):
Alkylating agents add ethyl or methyl
g p
groups
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Fig. 7.10b
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How mutagens alter DNA:
Chemical action of mutagen (cont)
Alter base structure and
properties (cont):
Deaminating agents
remove amine (-NH
( NH2)
groups
Fig. 7.10b
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How mutagens alter DNA:
Chemical action of mutagen (cont.)
Insert between bases: Intercalating
agents
Fig.
g 7.10c
Fig.27
7.10b
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DNA repair mechanisms that are
very accurate
Reversal of DNA base alterations
Homology-dependent repair of damaged bases
or nucleotides
• Base excision repair (Fig 7.11)
• Nucleotide excision repair (Fig 7.12)
Correction of DNA replication errors
• Particularly important
for removing uracil
(created by cytosine
deamination) from DNA
Fig. 7.11
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Nucleotide excision repair corrects
damaged nucleotides
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In bacteria, methylmethyl-directed mismatch
repair corrects mistakes in replication
• Parental DNA strand marked by
adenine methylase
• UvrA – UvrB complex
scans for
f distortions
di t ti
to
t
double helix (e.g.
th i di
thymine
dimers))
ƒ Methyl group added to A in GATC
sequence
q
ƒ Newly-replicated DNA isn't yet
methylated
• UvrB – UvrC complex
nicks the damaged
DNA
Fig. 7.12
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ƒ 4 nt to one side of damage
ƒ 7 nt to the other side of
damage
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• Different glycosylases
cleave specific
g bases
damaged
• Alkyltransferase – removes alkyl groups
• Photolyase – splits covalent bond of thymine dimers
• Methyl-directed mismatch repair (Fig 7.13)
Base excision repair
removes
damaged bases
• MutS and MutL bind to mismatched
nucleotides
• MutH nicks the unmethylated
strand
t d opposite
it the
th methylated
th l t d
GATC
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Fig. 7.13
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DNA repair mechanisms that are
error--prone
error
In bacteria, methylmethyl-directed mismatch
repair corrects mistakes in replication
SOS system – bacteria
• Gap made in
unmethylated
th l t d ((new))
strand by DNA
exonucleases
l
• Gap filled in by
DNA polymerase
using the
methylated (old)
strand as template
• Used at replication forks that stalled because of
unrepaired DNA damage
Sloppy DNA polymerase used instead of normal
• "Sloppy"
polymerase
• Adds random nucleotides opposite damaged bases
Nonhomologous
g
end-joining
j
g ((Fig
g 7.14))
Fi 7.13
Fig.
7 13 (cont)
(
t)
• Deals with double-strand DNA breaks caused by Xrays or reactive oxygen
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Repair of double
double--strand breaks by
nonhomologous endend-joining
Unrepaired doublestrand
t d breaks
b k can
lead to lethal
chromosome
h
rearrangements (e.g.
d l ti
deletions,
iinversions,
i
translocations)
Resection step can lead
to loss of DNA
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Health consequences of mutations
i genes encoding
in
di
DNA repair
i
enzymes
• Xeroderma pigmentosum:
• Mutations in one of seven
genes encoding
di enzymes
involved in nucleotide
excision
i i repair
i
Fig. 7.15
Hereditary forms of colorectal cancer (not shown):
Mutations in human homologs of bacterial genes (MutS and
MutL) involved in mismatch repair
Fig. 7.14
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C
Consequences
off mutations
t ti
The Ames test for
carcinogens using
his- mutants of
Salmonella
typhimurium
• Germ line mutations – passed on to
nextt generation
ti and
d affect
ff t th
the evolution
l ti
of species
• Somatic mutations – affect the survival
of an individual
More sensitive:
Second mutant
Æ nucleotide
excision
i i system
Third mutant
Æ cell wall
• Cell cycle mutations may lead to cancer.
• Because of potential harmful affects of
mutagens to individuals, tests have
been developed to identify carcinogens
carcinogens.
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Fig. 7.16
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Drosophila eye color mutations
produce a variety of phenotypes
What mutations tell us about gene
structure
Complementation testing
• Reveals whether two mutations are in a single gene or
i diff
in
differentt genes
• "Complementation group" is synonymous with a gene
Do these phenotypes result from allelic
mutations or from mutations in different
genes?
Fine structure mapping
• Seymour Benzer used phage T4 mutants
p
evidence that a g
gene is a linear sequence
q
• Experimental
of nucleotide pairs
• Some regions of chromosomes have "hot spots" for
mutations
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Fig. 7.17
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C
Complementation
l
t ti ttesting
ti
Five complementation groups (different genes) for eye color.
Recombination mapping demonstrates distance between genes and alleles.
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Fig. 7.18
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Lectured
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Fig. 7.18
b,c by Han-Jia Lin
Lectured
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A gene is a linear sequence of
nucleotide pairs.
B
Benzer’s
’ experimental
i
t l procedure
d
• Seymore Benzer mid 1950s – 1960s
• If a gene is a linear set of nucleotides
nucleotides,
recombination between homologous
chromosomes carrying different mutations
within the same gene should generate
wild type
wild-type.
• T4 phage as an experimental system:
• Can examine a large number of progeny to
detect rare mutation events
• Could allow only recombinant phage to
proliferate while parental phages die
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• Generated 1612 spontaneous point mutations
and some deletions
• Mapped location of deletions relative to one
another using
g recombination
• Found approximate location of individual
point mutations by
p
y deletion mapping
pp g
• Then performed recombination tests between
all p
point mutations known to lie in the same
small region of the chromosome
p of the rII g
gene
• Result – fine structure map
locus
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How recombination within a gene could
generate wild-type
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Fig. 7.19
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W ki with
Working
ith T4 phage
h
Fig. 7.20 a
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Complementation test to for mutations
in different genes
Ph
Phenotpyic
t i properties
ti off T4 phage
h
Fig. 7.20 b
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Fig.
7.20 c
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Detecting recombination between two
mutations in the same gene
Deletions for rapid mapping of point mutations to a
region of the chromosome
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Fig. 7.20
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Fig. 7.21 a
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Fi structure
Fine
t
t
map off rII
II gene region
i
Recombination
mapping to identify the
location of each point
mutation within a small
region
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Fig. 7.21
b by Han-Jia Lin
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Mutation hotspots suggest
that some nucleotides are
more susceptible
tibl tto
mutations than others
Fig. 7.21 c
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What mutations tell us about gene
function
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Alk
Alkaptonuria:
i A
An iinborn
b
error off metabolism
b li
Garrod (1902) – some human diseases result from
"inborn errors of metabolism" ((Fig
g 7.22))
Beadle and Tatum (1940s) – "the one gene, one
enzyme" hypothesis (Fig 7.23)
enzyme
• Neurospora crassa, mutants in arginine (arg) synthesis
• Genetic dissection of a biochemical pathway
Ingram (mid-1950s) – mutations in a gene can
result
lt iin amino
i acid
id substitutions
b tit ti
th
thatt di
disruptt th
the
function of the encoded protein
• Missense substitution in hemoglobin causes sickle cell
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anemia
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Fig 7.22
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Beadle and Tatum – One gene, one
enzyme
Experimental support
for the 䇾one gene,
g
,
one enzyme䇿
enzyme䇿
hypothesis
• 1940s – isolated mutagen induced mutants
that disrupted synthesis of arginine
arginine, an amino
acid required for Neurospora growth
• Auxotroph – needs supplement to grow on
minimal
i i l media
di
• Prototroph – wild-type that needs no supplement;
can synthesize all required growth factors
• Recombination analysis located mutations in
four distinct regions of genome.
• Complementation tests showed each of four
regions correlated with different
complementation group (each was a different
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gene).
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Scheme
S
h
used
d by
b
Beadle and Tatum
for isolation of arg –
auxotrophs in
Neurospora
Fig. 7.23 a
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Interpretation of Beadle and Tatum
experiments
• G
Growth
th response
if nutrient is added
to minimal
medium
• Inferred
biochemical
pathway
• Each gene controls the synthesis of an
enzyme involved in catalyzing the
conversion of an intermediate into
arginine.
• Each ARG gene
encodes an
enzyme needed to
convert one
intermediate to the
next in the p
pathway
y
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Fig. 7.23
b-c by Han-Jia Lin
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Genes specify the identity and order of
amino acids in a polypeptide chain.
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N terminus of a protein contains a free amino group.
C terminus of protein contains a free carboxylic acid group.
• Proteins are linear polymers of amino acids
linked by peptide bonds.
• 20 different amino acids are building blocks of
proteins.
• NH2-CHR-COOH – carboxylic acid is acidic, amino
group is basic.
• R is the side chain that distinguishes each amino
acid.
Fig. 7.24 a
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Fig. 7.24
c by Han-Jia Lin
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R is the side group that distinguishes each amino
acid.
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Fig. 7.24
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Fig. 7.24
b, cont.
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Genes specify the amino acid sequence of a
polypeptide – example, sickle cell anemia.
Mutant E chain of
hemoglobin forms
aggregates that
cause red blood
cells to sickle.
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Fig. 7.24
b, cont.
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Fig. 7.25
a by Han-Jia Lin
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Sequence of amino acids determine a protein’s
primary, secondary, and tertiary structure.
Si
SickleSickle
kl -cell
ll anemia
i is
i pleiotropic
l i t
i
Fig 7.25b
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Fig. 7.26
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Some proteins are multimeric, containing subunits
composed of more than one polypeptide.
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How do genotypes and phenotypes
correlate?
• Alteration of amino acid composition of
a protein
• Alteration of the amount of normal
protein produced
• Changes
Ch
iin diff
differentt amino
i acids
id att
different positions have different effects.
• Proteins have active sites and sites
involved in shape or structure.
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Fig. Lectured
7.27 by Han-Jia Lin
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