Download Ch8 BacterialgeneticsPrt2HO.ppt

10/23/13
Chapter 8: Bacterial Genetics
Genetic changes in
bacteria occur via:
-mutations
-gene transfer
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What are mutations?
•  Change in the base sequence of the
DNA
•  Do they always change the genetic
code?
Causes of mutations in
bacteria
• 
• 
• 
• 
Most are spontaneous
Errors made by DNA Polymerase
UV light exposure
Chemical damage of bases in DNA
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Types of Mutations
•  Base-pair mutation
–  Missense mutation
–  Nonsense mutation
–  Silent mutation
Base-pair mutations
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5′
• • • • • •AT G
•
• • • • • C
3′
T
A
3′
• • • •• •
• • • • • •5′
DNA
T G T
A C A
T G C
A C G
T G G
A C C
T G A
A C T
Mutation
UG U
UG C
UG G
UG A
Transcribed codon
Cysteine
Cysteine Tryptophan Stop codon Amino acid translated
Wild type
Silent
mutation
Missense
mutation
Nonsense
mutation
Outcome
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Types of Mutations
• 
•  Frameshift
Changes the
reading frame
Wild type
5′
3′
3′
C G G T A CG T T A A A
G C C A T GCA A T T T
DNA
5′
5′
3′
C G G
Deletion or addition of nucleotides
–  Impact depends on number of
nucleotides
–  Three pairs changes one codon
•One amino acid more or less
•One or two pairs yields
frameshift mutation
U A C
Arginine Tyrosine
GU U
A A A
Transcribed codons
Valine
Lysine
Amino acids translated
Mutant
Base pair
addition
3′
5′
- Different set of codons translated
- Often results in premature stop
codon
•  Shortened, nonfunctional
protein
•  Knockout mutation
3′
5′
CGGA T A CG T T A A A
G C C T A T G C A A T T T
DNA
5′
3′
C G G
Arginine
A U A
C G U
U A A
Isoleucine Arginine
Stop
Transcribed codons
Amino acids translated
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5′
Base-pair mutation:
missense
3′
3′
5′
DNA strand
separation
DNA
replication
5′
5′
3′
3′
5′
DNA replication, an incorrect
nucleotide is incorporated
3′
5′
3′
3′
5′
Wild type
3′
5′
Base substitution
5′
DNA
replication,
generating
a mutation
5′
3′
Mutant
3′
DNA strand
separation
3′
5′
DNA replication
3′
3′
5′
5′
5′
3′
Wild type
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DNA Repair Can Prevent Mutations
Figure 8.2
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DNA Repair Can Prevent Mutations
Repair
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Figure 8.2
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DNA Repair Can Prevent Mutations
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Figure 8.2
What can cause mutations?
•  Chemicals (nitrous acid)
•  Physical mutagens (uv light)
•  Biological mutagens (transposons)
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Barbara McClintock: jumping
genes biological mutagen
Transposons
Can move from one location to another
This jumping around is called
transposition
Genes are inactivated
Function destroyed
Most transposons have transcriptional
terminators---Blocks expression of
downstream genes
•  Induced mutations result from outside
influence
–  Agent that induces change is mutagen
–  Geneticists may use mutagens to increase
mutation rate
–  Two general types: chemical, radiation
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•  Chemical mutagens may cause base
substitutions or frameshift mutations
•  Some chemicals modify nucleobases
–  Change base-pairing properties
–  Increase chance of incorrect nucleotide
incorporation
•  Nitrous acid (HNO2) converts cytosine to uracil
–  Base-pairs with adenine instead of guanine
Nitrous acid as a chemical
mutagen
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Alkylating agents add alkyl groups onto
nucleobases
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Hydrogen bonds formed
with complementary bases
O
O
H
N
N
N
H
N
deoxyribose
N
H
N
Alkylating agent N
H
deoxyribose
Guanine
(pairs with C)
Nitrosoguanidine adds
methyl group to
guanine
N
H
H
N
Added
alkyl group
CH3
Base-pairs with thymine
N
H
Methylguanine
(sometimes pairs with T)
(a)
Wild type
3′
AGT
T CA
3′
5′
5′
Nitrosoguanidine
treatment
5′
3′
A G *T
T C A
3′
DNA
replication
5′
Guanine (G) is converted
to methylguanine (G*)
5′
3′
A G*T
T T A
3′
5′
DNA
replication
Mutant
5′
3′
A A T
T TA
3′
5′
Thymine (formerly paired
Methylguanine of template
strand pairs with thymine (T) with G*) now serves as
template and pairs with
instead of cytosine (C)
adenine (A)
(b)
Nucleoside analogs are mutagens
mistaken identity
Base analogs resemble
nucleobases
-different hydrogenbonding properties
- incorporated by DNA
polymerase
5-bromouracil resembles
thymine, (pairs with
cytosine)
2-amino purine resembles
adenine, can pair with
cytosine
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Intercalating agents
cause frameshift mutations
Flat molecules that insert
between the base pairs in
DNA strand
Pushes nucleotides apart,
produces space
Causes errors during replication
UV light as a mutagen
X rays cause single- and double-strand breaks in
DNA-can produce lethal deletions
X rays can alter nucleobases
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Repair mechanisms
•  Wrong nucleotide inserted
–  Proofreading
–  Mismatch repair
Repair of Damaged DNA
•  Enormous amount of spontaneous and
mutagen-induced damage to DNA
–  If not repaired, can lead to cell death; cancer in
animals
•  E.g., in humans, two breast cancer susceptibility
genes code for DNA repair enzymes; mutations in
either result in 80% probability of breast cancer
–  Mutations are rare; alterations in DNA generally
repaired before being passed to progeny
–  Several different DNA repair mechanisms
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Repair of Errors in Nucleotide Incorporation
–  Mismatch Repair
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•  Fixes errors missed by DNA
polymerase
•  Enzyme cuts sugar-phosphate
backbone
•  Another enzyme degrades short
region of DNA strand
•  Methylation of DNA indicates
template strand
–  Methylation takes time, so newly
synthesized strand is unmethylated
•  DNA polymerase, DNA ligase
make repairs
Template strand
1
3′
CH3
CH3
5′
3′
C T A A G C T G A G
G A T T T G A C T C
5′
The wrong nucleotide
is incorporated during
DNA synthesis.
Newly synthesized
strand
2
3′
CH3
CH3
5′
3′
C T A A G C T G A G
G A T T T G A C T C
5′
Near the site of the mismatched
base, an enzyme cuts the
sugar-phosphate backbone
of the unmethylated strand.
Cut
3
CH3
5′
CH3
C T A A G C T G A G
G A
C T C
3′
5′
3′
5
3′
C T A A G C T G A G
G A T T C G AC T C
CH3
5′
CH3
5′
3′
An enzyme degrades a
short stretch of the strand
that had the error.
CH3
CH3
4
3′
5′
C T A A G C T G A G
G AT T C G A C T C
DNA ligase
3′
5′
DNA polymerase synthesizes
a new stretch, incorporating
the correct nucleotide.
DNA ligase joins the 3′ end
of the newly synthesized
segment to the original
strand.
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Template strand
1
CH3
CH3
5′
Mismatch Repair
3′
3′
C T A A G C T G A G
G A T T T G A C T C
5′
The wrong nucleotide
is incorporated during
DNA synthesis.
Newly synthesized strand
2
5′
3′
CH3
CH3
3′
C T A A G C T G A G
G A T T T G A C T C
5′
Near the site of the mismatched
base, an enzyme cuts the
sugar-phosphate backbone
of the unmethylated strand.
Cut
3
CH3
5′
CH3
C T A A G C T G A G
G A
C T C
3′
5′
3′
5
3′
C T A A G C T G A G
G A T T C G A C T C
CH3
5′
CH3
5′
3′
5′
An enzyme degrades a
short stretch of the strand
that had the error.
CH3
CH3
4
3′
C T A A G C T G A G
G A T T C G A C T C
DNA ligase
3′
5′
DNA polymerase synthesizes
a new stretch, incorporating
the correct nucleotide.
DNA ligase joins the 3′ end
of the newly synthesized
segment to the original
strand.
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Photoreactivation
Repair of
UV damage
Covalent bonds
5′
3′
G C G A T T G A CG
C G C T AA C T G C
3′
G C G A T T G A CG
C G C T A A C T GC
Thymine dimer distorts the
DNA molecule.
5′
3′
5′
•  Two repair
mechanisms
3′
5′
An enzyme uses visible light to
break the covalent bond of the
thymine dimer, restoring the DNA
to its original state.
Excision repair
Covalent bonds
–  Light repair
–  Dark repair
5′
3′
G C G A TT G A CG
C G C T AA C T G C
Cut
3′
3′
G C
CG
C G C T A A C T GC
5′
3′
Thymine dimer distorts the
DNA molecule.
5′
Cut
A T TGA
G
5′
3′
5′
3′
G C G A T T G A CG
C G C T A A C T GC
5′
An enzyme removes the
damaged section by cutting
the DNA backbone on either
side of the thymine dimer.
The combined actions of DNA
polymerase and DNA ligase fill in
and seal the gap.
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Repair of
oxidation
damage
1
5′
3′
2
A G– O C
T C G
T
A
5′
3′
C
G
T
A
A
T
A
T
C
C
G
T
A
5′
DNA contains oxidized
guanine (G–O) as a result
of oxidation damage.
Glycosylase removes the
oxidized nucleobase from the
sugar-phosphate backbone.
Cut
3′
C
G
T
A
A
T
C
G
T
A
T
C T
G A
A
T
A
T
C
C
G
T
A
T
C
C
G
T
A
A
T
G
C
C
G
T
A
5′
3′
5
A
T
5′
3′
4
T
A
5′
3′
3
3′
C
G
5′
3′
5′
3′
5′
3′
5′
At the site of the missing
nucleobase, an enzyme cuts the
sugar-phosphate backbone.
DNA polymerase degrades a
short stretch of the strand.
The combined actions of DNA
polymerase and DNA ligase
fill in and seal the gap.
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Repair of Thymine Dimers
•  Several methods to repair damage from UV
light (continued…)
–  SOS repair: last-ditch repair mechanism
•  Induced following extensive DNA damage
•  Photoreactivation, excision repair unable to correct
•  DNA and RNA polymerases stall at unrepaired sites
•  Several dozen genes in SOS system activated
–  Includes a DNA polymerase that synthesizes even in
extensively damaged regions
–  Has no proofreading ability, so errors made
–  Result is SOS mutagenesis
Why use bacteria to study
mutations?
•  Only have one chromosome…one copy
of each gene
•  Easy to grow
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Direct selection
•  Testing for traits that are easily
identified
–  Colony color
–  Motility
–  Resistance to antibiotics
Indirect selection
•  A way to look at traits that are not easy
to detect
–  For example, changes in metabolism
•  Replica plating
–  A way to identify AUXOTROPHS from
PROTOTROPHS
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Replica Plating: indirect
selection
Penicillin enrichment of
mutants
Increases proportion of
auxotrophs in broth culture
Penicillin kills fast growing
cells, the prototrophs)
The auxotrophs survive.
Penicillinase is then added.
Cells plated on rich medium
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Testing chemicals for mutagenicity…Ames test
Mutant Selection
–  Ames test measures effect of chemical on
reversion rate of histidine-requiring Salmonella
auxotroph
•  Uses direct selection
•  If mutagenic, reversion rate increases relative to
control
•  Rat liver extract added since non-carcinogenic
chemicals often converted to carcinogens by animal
enzymes
•  Additional tests then conducted on mutagenic
chemicals to determine if carcinogenic
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