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723
MCB 3020, Spring 2005
Chapter 10:
Microbial Genetics I
Mutations
Genetics :
the study of the mechanisms of
heredity and variation in organisms
DNA
DNA
724
RNA
protein
Central dogma
phenotype
725
A. The genotype determines the
possible phenotypes of an organism.
Genotype
the exact genetic composition
(DNA sequence) of an organism
Phenotype
the observable characteristics
of an organism
B. Why study prokaryotic genetics?
726
1. Prokaryotes are relatively simple:
haploid, easy to grow
2. Many principles of genetics are the
same in prokaryotes and eukaryotes.
3. Molecular cloning and biotechnology
4. Control of pathogenic microorganisms
TB
Life evolves. This leads to diversity.
727
extant species
ancestral cell
mutation,
DNA transfer
and natural selection
(evolutionary time)
It is likely that all organisms are related to
a single ancestral cell or group of cells. TB
D. Genetic diversity can result from
1. Mutations
2. DNA transfer
728
Molecular Genetics I: Mutations
729
I. Mutations
II. Effects of mutations on protein structure
III. Effects of mutations on protein function
IV. Effects of mutations on phenotype
V. Mutagens
I. Mutations
inheritable changes in the
genotype (DNA sequence) of
an organism
730
Mutations can play an important role
in genetic diversity and evolution.
731
A. A base pair change is an example
of a mutation.
...GATCGGATC...
...CTAGCCTAG...
mutation
...GATAGGATC...
...CTATCCTAG...
TB
B. Mutations can lead to biological
variation
732
Most mutations are harmful or neutral.
Rare beneficial mutations and natural
selection lead to new species.
TB
C. Most mutations result from DNA
replication errors.
733
DNA polymerases sometimes make
mistakes that are not repaired.
DNA damage increases the
likelihood of such mistakes.
TB
DNA damage can lead to mutation, but is not734
a mutation per se because it is not heritable.
...GATCGGATC...
...CTAGCCTAG...
DNA damage (alkylation)
...GATCGGATC...
...CTAGCCTAG...
CH3
methyl-G
TB
735
D. Mutation frequencies are thought to
be roughly similar in all organisms:
-9
~10
to
-10
10 /
base pair / generation
Thus, in general, mutations are rare.
TB
736
E. Mutants can be derived from wild-type
strains (or from other mutant strains).
Wild-type: the original strain
of an organism isolated from
mutation
nature
Mutant: an organism with
a genome that carries a mutation
mutation
Mutations in genes that encode
proteins can affect
737
• protein structure
• protein function
• the phenotype of the organisms
738
II. Effects of mutations on protein structure
A. base pair changes
1. silent mutations
2. missense mutations
3. nonsense mutations
B. deletions
C. insertions
D. frameshift mutations
E. inversions
F. duplications
Overview: Effect of mutations on
protein structure
mutation
739
gene
transcription
mRNA
translation
OR
Translation has two possible outcomes:
(1) a change in the amino acid sequence, or
TB
(2) no change.
740
A. Base-pair changes (point mutations)
A heritable change in a
single base pair of DNA
1. silent mutations
2. missense mutations
3. nonsense mutations
741
1.Silent mutations
DNA
...TAC... mutation ...TAT...
...ATG...
...ATA...
RNA
...UAC...
...UAU...
tyrosine
tyrosine
Polypeptide
No change in the polypeptide
TB
2. Missense mutations
742
DNA
...TAC... mutation ...AAC...
...ATG...
...TTG...
RNA
...UAC...
...AAC...
tyrosine
asparagine
Polypeptide
One amino acid is changed in the polypeptide. TB
3. Nonsense mutations
(mutation results in a stop codon)
743
DNA
...TAC... mutation ...TAG...
...ATG...
...ATC...
RNA
...UAC...
...UAG...
tyrosine
stop codon
Polypeptide
A truncated polypeptide is made.
TB
B. Deletions
One or more base pairs are lost
744
ATGAAAGAG....
ATGGAG....
Possible results
a. amino acids or polypeptides can be lost
b. frameshifts can occur (see below)
TB
C. Insertions
745
One or more base pairs are gained
ATGGAG....
ATGAAAGAG....
Possible results
a. amino acids or polypeptides can be gained.
b. frameshifts can occur (see below)
TB
D. Frameshift mutations
746
Insertions or deletions that change
the translational frame
ATGCAAGTTG....
one base pair deletion
ATGAAGTTG....
Two changes in polypeptides are possible:
(1) every amino acid downstream of the mutation is changed,
(2) a truncated (shortened) protein is produced.
TB
DNA can have 3 reading frames:
747
AT G C AA G T T G A
AT G C AA G T T G A
#1
A T cys
G C A AlysG T TleuG A
#2
AT G C AA G T T G A
#3
met
gln
ala
val
ser
STOP
Frameshift mutations change the
translational reading frame.
748
ATGCAAGTTGA….
met
gln
val
one base pair deletion
ATCAAGTTGA
ile
lys
leu
(Frameshifts occur only if
insertion or deletion is in the
reading frame section of a
protein-encoding gene.)
749
E. Inversions
chromosomal segment is inverted
...ATGGAAGAG....
...TACCTTCTC....
...ATTTCCGAG....
...TAAAGGCTC....
A number of changes in polypeptides are possible.TB
750
F. Duplications
chromosomal segment is duplicated
...ATGGAAGAG....
...TACCTTCTC....
...ATGGAAGGAAGAG....
...TACCTTCCTTCTC....
A number of changes in polypeptides are possible.TB
III. Effects of mutations on protein
function
751
1. No effect (common)
2. Loss of function (common)
3. Partial loss of function (leaky)
4. Conditional loss of function
5. Change of function (rare)
6. Restoration of function (reversion)
TB
1. No effect (common)
752
Depending on the protein, up to
80% of the amino acids may only
function as spacers.
wild type
(normal protein)
mutant protein
TB
2. Loss of function (common)
Examples:
753
a. A change in an amino acid that
participates directly in catalysis
(change in the active site)
wild type
(normal protein)
mutant
protein
TB
2. Loss of function (contd.)
754
b. A change in an amino acid that
causes the protein to misfold.
wild type
protein
misfolded
mutant
protein
degradation
amino acids
TB
3. Partial loss of function,
"leaky" (common)
755
Reduction in the catalytic activity
of an enzyme due to a change in
3-D shape, and / or stability.
wild type
(normal)
protein
mutant
protein
TB
4. Conditional loss of function (common)
756
e.g. Temperature-sensitive (heatsensitive) mutations.
42°C
30°C
mutant
protein
properly
folded
misfolded
degradation
amino acids
TB
5. Change of function (rare)
757
e.g. change in specificity
wild type
protein
converts maltose
to 2 glucose
mutant
protein
converts lactose
to glucose and galactose
TB
6. Restoration of function, reversion
("back mutation") (rare)
mutant
protein
758
nonfunctional
a second mutation
functional
TB
a. Same site revertants
i. true revertants
759
A second mutation restores the
original DNA sequence.
ii. others
A second change at the same site
results in a less harmful amino acid
change, or the original amino acid.
TB
760
b. Second site revertants (suppressors)
i. intragenic
A second mutation at a different site
within the same gene restores function.
ii. intergenic
A second mutation in a different gene
restores function.
TB
Example of an intragenic suppressor
+
Lys 121
_
Asp 44
761
a salt bridge between
Lys 121 and Asp 44 is
essential to protein
folding.
A mutation that converts Lys 121 to Glu
destroys protein activity.
A second mutation that converts Asp 44
to His restores protein activity.
Note that Asp and Glu are negatively charged
and that Lys and His are positively charged. TB
IV. Effects of mutations on phenotype
762
Phenotype
The observable characteristics
of an organism
Mutations can have many
different effects on phenotype.
TB
A. Loss of enzyme activity
Examples:
763
1. If a mutations destroys an enzyme needed
for pigment formation, an albino can result.
2. If a mutation inactivates an enzyme for
lactose catabolism, a microbe unable to
grow on lactose will result.
TB
B. Loss of regulatory proteins
764
1. inability to induce enzymes
2. inability to differentiate
3. inability to tax toward nutrients
etc. etc. etc.
C. Loss of structural proteins
D. Mutations in tRNA or rRNA
TB
V. Mutagens
765
Substances that increase
mutation frequency.
In the lab, mutagens can be
used to create mutations
for genetic analysis.
TB
A. Mutation frequencies
766
Spontaneous mutations occur
with a frequency of about
-9
10 / base pair / generation
Mutagens are used to increase
mutation frequencies to
-6
-7
~10 to 10 / base pair / generation
-3
-4
(~10 to 10 / gene / generation). TB
B. Types of mutagens and the
mutations they cause.
767
1. Base analogs
Compounds structurally similar
to the normal DNA bases
TB
Thymine
Bromouracil
O
O
H
O
CH3
N
N
H
H
O
768
Br
N
N
H
• Bromouracil will be incorporated into DNA
in place of thymine.
• During DNA replication, bromouracil can
mispair with guanine and cause point
TB
mutations.
2. Alkylating agents
Compounds that chemically
modify DNA bases via alkylation
769
During DNA replication modified
bases mispair causing single base
pair change (point) mutations.
Example: dimethyl sulfate
TB
3. Intercalating agents
770
Chemicals that insert between DNA base pairs.
H-bonds
backbone
DNA bases
...
...
...
...
...
...
intercalating
agent
(ethidium
bromide)
Intercalating agents lead to small deletions
TB
and insertions during DNA replication.
4. Radiation
771
Ultraviolet light (UV)
H
O
O
N
N
CH3
CH3
O
N
N
H
O
Thymine dimer: two "T"s on the same strand
become covalently bonded.
Thymine dimers lead to various replication errors.
TB
1. Understand how genotype affects phenotype.
772
2. Define mutation. Understand the role of mutations in genetic diversity and
evolution. Is chemical modification of a DNA base considered a mutation? why?
3. What is the most common cause of spontaneous mutations? What is the typical
mutation frequency in most organisms? Define wildtype and mutant.
4. What is a point mutation? Understand the effects of silent, missense and
nonsense mutations on protein primary structure.
5. Define deletions, insertions, frameshift mutations, inversions, and duplications.
Understand how these mutations influence protein structure.
6. Be able to distinguish between the different effects of mutations on protein
function. What are most common effects that mutations have on protein
function? Which are rare? Understand the terms leaky mutant, conditional loss
of function, temperature-sensitive mutations, back mutation, reversion,
revertants (know the different types), intragenic and intergenic suppression.
7. Describe how a mutation might change the substrate specificity of an enzyme.
8. In general how do mutations affect phenotype?
9. In genetics, what is main use of mutagens? How do they affect mutation freq?
Describe how base pair analogs, alkylating agents, intercalating agents and UV
radiation lead to mutations. Know the examples! What is a thymine dimer?
773
MCB 3020, Spring 2005
Chapter 10:
Microbial Genetics II
Genetics Techniques
Molecular Genetics II: Techniques
I. The isolation of mutants
II. The Ames' test
III. General recombination
IV. Complementation
774
Some laboratory uses of mutations
775
1. Mutations can help identify genes
involved in particular biological
processes.
2. Mutations can help to determine the
function of specific genes.
(e.g. metabolic pathway genes,
regulatory genes, transport genes)
Some advantages of mutant
studies with bacteria
776
Bacteria are haploid.
Bacteria are easily grown
in large numbers.
TB
I. The isolation of mutants
777
To use mutations to identify genes
and their functions, the first step is to
create and isolate organisms with
mutations that affect the process of
interest (e.g. histidine biosynthesis).
Because specific mutations are
relatively rare, procedures need to
be efficient.
TB
A. Isolation of histidine biosynthetic
mutants in Escherichia coli
778
(strains with mutations in his biosynthetic genes)
1. Designate a particular E. coli strain
as the wildtype strain (His+ phenotype).
2. Grow a broth culture of the wildtype.
TB
3. Treat culture with mutagen (produces 779
mutations at random locations).
mutagenized culture
dilute and plate
on a rich medium
TB
4. Select or screen for mutant strains
that require histidine for growth
(His- phenotype).
Replica plating
minimal medium
without histidine
minimal medium
with histidine
780
Replica plating
781
Simultaneous transfer of all colonies on
master plate to several different media.
minimal
medium
minimal
medium + histidine
Between 1/1000 and 1/10,000 colonies will have
a mutation in a particular gene.
TB
5. Prepare pure cultures from strains
that require histidine for growth.
782
6. These strains have mutations in
genes needed for histidine
biosynthesis.
7. Write down a list of mutant strains
that indicates their genotype and
phenotype.
TB
In practice, the genotype and phenotype 783
of the mutant strains is indicated as their
differences from the wildtype strain.
Strain #
1
2
genotype
his-1
his-2
phenotype
HisHis-
Eventually the dash replaced by a
letter designating a specific gene
TB
Naming genes and mutations
784
(Example for a histidine biosynthetic gene)
Gene
hisC
Mutant genes hisC1, hisC2, hisC3
Protein
HisC or (name of protein)
Phenotypes
His+ (can make histidine)
His- (cannot make histidine)
785
Auxotrophs
• A His- mutant of E. coli cannot make histidine
and requires histidine as a growth factor
• An E. coli mutant with a His- phenotype
is a histidine auxotroph.
• It will grow in the presence, but not
in the absence, of histidine.
• Auxotroph: a nutritional mutant that has
a requirement for a growth factor (relative
to its parent strain, the prototroph)
B. How could you isolate mutants
in lactose catabolism?
786
Use the same procedure as above,
but screen for mutant strains
unable to catabolize lactose.
A convenient screen for lactose
catabolism is the MacConkey
indicator medium.
TB
MacConkey lactose indicator medium
787
Detects acids produced from lactose
catabolism.
Lac+
Lac
lac mutants are unable
to produce acid and
hence are white.
Between 1/1000 and 1/10,000 colonies will have
a mutation in a particular gene.
TB
C. Screening and Selection
788
1. Screening
Identification of particular mutants by
comparing their properties to the wild
type (usually colony properties).
Examples
indicator plates (see above)
loss of pigmentation
TB
2. Selection
789
Identification of particular mutants by
using conditions that prevent the
growth of other cells.
e.g. selection for antibiotic resistance
Colonies that grow
are antibiotic-resistant
mutants
medium with
antibiotic
(plate ~108 cells)
TB
II. The Ames test
A test used to identify mutagens
1. Spread ~108 His- cells on
minimal plates (no histidine)
790
minimal
2. Soak filter disk with test compound
and place on plate.
3. Incubate plates and examine
(look for increase in # of back mutations
that restore His+ phenotype)
TB
Possible results
control
791
test compound test compound
#1
#2
revertants
is not a
mutagen
is a
mutagen
TB
III. General recombination
792
DNA rearrangements involving
crossovers between homologous
DNA sequences.
TB
A. Cellular uses of recombination
793
1. The generation of genetic variation
in eukaryotes during meiosis
2. The generation of variation in
prokaryotes via its role in gene
transfer
3. DNA Repair
TB
B. Genetic crossovers
794
DNA exchanges used in recombination
x
"x" is the crossover site
breakage of
phosphodiester bonds
TB
795
reunion of
phosphodiester bonds
For general recombination, crossovers only
occur between between homologous (identical
or nearly identical) DNA segments.
TB
C. Outcomes of recombination
1. identical
sequences
A
B
796
C
X
A
B
C
A
B
C
A
B
C
single crossover
identical
sequences
TB
2. nearly
identical
sequences
A
B
C
797
X
a
B
c
a
B
C
A
B
c
single crossover
recombinant
sequences
TB
X
3. Two circular DNA molecules
798
single crossover
integration
TB
4. circular + linear
799
XX
double crossover
TB
5. single strand exchange
800
TB
D. A model for recombination
801
alignment of
homologous
sequences
nicking
TB
802
unwinding (RecBCD)
strand invasion (RecA)
TB
803
strand exchange
From this point there are two
main methods of resolution.
TB
1. Inner strand resolution
(break and religate inner strands)
804
inner
break
TB
805
religate
TB
2. Outer strand resolution
(break and religate outer strands)
break
806
outer strands
TB
d
c
b
a
a
b
c
d
807
religate
a
b
a
b
c
d
c
d
TB
IV. Complementation
808
In a cell that has a recessive mutation,
restoration of the wild type phenotype
by a second DNA molecule.
A
A
TB
809
A. In laboratory research,
complementation has important uses.
1. Screening for clones of interest.
2. Verification that a particular mutation
results in a particular phenotype
TB
B. How complementation is observed 810
1. Start with a bacterium with a recessive
mutation resulting in Trp– phenotype
or any other phenotype.
chromosome
Mutation 1
A
TB
2. Introduce a second DNA molecule into the 811
bacterium by one of several methods.
mutation 1
second DNA
molecule
(plasmid)
A
A
Restoration of the wildtype (in this case Trp+)
phenotype is termed complementation.
TB
C. Key Point
812
Complementation indicates that the
second DNA molecule (the plasmid)
has a good copy of the chromosomal
gene that is mutated.
TB
Study objectives
813
1. Describe two ways that mutations are used in laboratory research.
2. Describe how bacterial biosynthetic and catabolic mutants can be isolated.
What is replica plating?
3. Know how to write the names of genes, mutant genes, protein products, and
phenotypes. Pay particular attention to font and capitalization.
4. What are prototrophs and auxotrophs?
5. Compare and contrast screens and selections. Are screens and selections
equally useful for the isolation of very rare mutations?
6. Understand what the Ames test is used for and how it works.
7. Name three cellular uses of general recombination.
8. What are the constraints of genetic crossovers in general recombination?
9. Describe recombination, its main steps, and the key enzymes involved.
I will NOT ask about inner strand versus outer strand resolution.
10. What is complementation and what are its uses? What is the role of plasmids
in complementation?
814
MCB 3020, Spring 2005
Chapter 10:
Microbial Genetics III:
DNA transfer
Life evolves. This leads to diversity.
815
extant species
ancestral cell
mutation,
DNA transfer
and natural selection
(evolutionary time)
It is likely that all organisms are related to
a single ancestral cell or group of cells. TB
Molecular Genetics III: DNA Transfer
I. DNA transfer in prokaryotes
A. transformation
B. transduction
C. conjugation
II. Transposable elements
816
I. DNA transfer in prokaryotes
817
A. transformation
B. transduction
C. conjugation
TB
DNA transfer in prokaryotes
818
The transfer of donor DNA to
the genome of a recipient cell.
TB
Uses of DNA transfer
819
1. In natural environments, DNA
transfer is used to generate
genetic variation.
2. In the lab, DNA transfer is used for
genetic mapping and the
construction of recombinant
organisms with particular genotypes.
TB
820
A. Transformation
Transfer of free DNA to a bacterial
genome.
free DNA
TB
free DNA
821
chromosome
recipient cell
general recombination
transformant
TB
822
free DNA (plasmid)
transformant
General recombination is not necessary
because plasmids have origins of replication
TB
1. Competence
823
The capacity of cells to take up free DNA.
a. Some cells are naturally competent
b. In some cells, competence can be
induced by chemical and physical
treatment
c. In most cells, competence can be
induced by electroporation.
TB
Electroporation
824
cells + DNA
- electrode
+ electrode
A brief electric pulse induces pores
in the cell envelope and free DNA enters
TB
B. Transduction
825
Transfer of DNA by viral particles
1. Generalized transduction
2. Specialized transduction
TB
1. Generalized transduction
826
In generalized transduction,
transducing particles formed by
packaging errors can contain DNA
from any part of the donor genome.
Generalized transduction
827
donor cell
chromosome
viral replication
many
viruses
a few transducing
particles containing part of
donor cell DNA (formed
by packaging errors) TB
828
recipient
chromosome
transducing
particle
recipient cell
general recombination
transductant
TB
2. Specialized transduction
chromosome
829
donor cell (lysogen)
prophage
(integrated
bacterial virus)
improper
excision
viral
replication
transducing
particles TB
830
recipient cell
transducing
virus
chromosome
general
recombination
phage
integration
transductants
TB
Key points for specialized transduction
Transducing viruses formed by
improper excision can only transfer
DNA adjacent to the prophage
insertion site.
831
Transducing viruses can become part
of the recipient genome by general
recombination or integration.
TB
C. Conjugation
832
Direct cell to cell DNA transfer
involving certain plasmids.
Picture
7
TB
1. Conjugative plasmids
833
plasmids that mediate their own transfer
e.g. F-plasmid
tra
IS
IS
oriT
oriS
TB
2. The DNA transfer process
donor cell (F+)
F- pilus
F plasmid
chromosome
834
recipient cell (F-)
chromosome
The F-pilus is used for cell-cell attachment
TB
835
replication and
transfer of ssDNA
exconjugant
F+
F+
Note that the recipient cell becomes F+ TB
3. The F-plasmid can integrate
into the bacterial chromosome.
chromosome
Hfr strain
836
F plasmid
integration
integrated
F plasmid
TB
Integrated F plasmids can transfer the chromosome.
a. Hfr strain
A cell that carries the integrated
F plasmid is called the "Hfr" strain
Hfr stands for "high frequency of
recombination"
837
Hfr strain
A bacterium with an F plasmid
integrated into its chromosome.
Hfr strain
838
integrated
F plasmid
In nature, they can transfer genes and
play a role in generating variation.
In the lab, they are used for genetic
mapping (determining gene location). TB
839
b. The integrated F plasmid can
mobilize the chromosome (i.e. move
part of the chromosome to another cell)
Hfr strain
integrated
F plasmid
TB
Bacterial genetic maps
maps that show the relative locations
of genes.
840
spoT
galKTE
X
hisGDCBHAFIE
~500-12,000 genes
TB
c. The integrated F-plasmid can excise
841
improperly, forming a plasmid with part of
the host chromosome. The resulting
plasmid is called F-prime (F').
Hfr strain
Improper
excision
integrated
F plasmid
F'
TB
F-prime (F'):
842
An improperly excised F plasmid containing
a segment of bacterial chromosomal DNA.
Hfr strain
improper
excision
F plasmid
F'
TB
F-prime (F')
part of F plasmid
843
segment of
bacterial
chromosome
Formed by improper excision of Hfr
Can transfer chromosomal genes
TB
844
III. Transposable elements
A. examples
1. insertion sequences
2. transposons
B. transposition
C. uses of transposable elements
TB
Transposable elements
845
DNA segments that can move from one
location to another; "mobile DNA"
IS
IS
transposable
element
host DNA
disrupted, nonfunctional gene
TB
IS
IS
transposable
element
846
Found as part of the genome of all
organisms carefully examined
Their function is uncertain.They may
simply be "selfish DNA".
In the lab, transposons are used to create
mutations.
TB
A. Examples of transposable elements847
1. Insertion sequences
IS2
tnp
inverted terminal repeat
tnp = transposase gene
transposase catalyzes transposition
TB
2. Transposons
848
Tn5
kan str ble
IS50L
tnp
IS50R
host genes
Transposons typically consist of host gene(s)
flanked by insertion sequences.
TB
B. Transposition
849
The movement of a transposable element
1. conservative
One element is
moved to another site.
TB
Transposition
850
2. replicative
One element is duplicated.
The first copy stays at the original site.
The second copy is found at another site.
TB
C. Uses of transposable elements
851
1. In the lab, transposon mutagenesis
2. In nature, function is not known.
Just selfish DNA?
TB
Study objectives
852
1. Know that DNA transfer can generate genetic variation and can be used to
construct recombinant organisms.
2. Know how competence affects transformation and how competence is
induced.
3. Know how DNA is transferred by transformation, transduction, and
conjugation. Be able to compare and contrast these types of gene transfer.
4. Know what Hfr strains and F-prime plasmids are.
5. Be able to compare and contrast transposons and insertion sequences.
6. Understand conservative and replicative transposition and know that
transposition causes mutations.