MCB 3020, Spring 2005

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MCB 3020, Spring 2005
Chapter 10 Microbial Genetics I Mutations
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Genetics the study of the mechanisms of
heredity and variation in organisms
DNA
Central dogma
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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
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B. Why study prokaryotic genetics?
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
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Life evolves. This leads to diversity.
It is likely that all organisms are related to a
single ancestral cell or group of cells.
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D. Genetic diversity can result from 1.
Mutations 2. DNA transfer
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Molecular Genetics I Mutations
I. Mutations II. Effects of mutations on
protein structure III. Effects of mutations on
protein function IV. Effects of mutations on
phenotype V. Mutagens
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I. Mutations
inheritable changes in the genotype (DNA
sequence) of an organism
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A. A base pair change is an example of a
mutation.
...GATCGGATC... ...CTAGCCTAG...
mutation
...GATAGGATC... ...CTATCCTAG...
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B. Mutations can lead to biological variation
Most mutations are harmful or neutral.
Rare beneficial mutations and natural selection
lead to new species.
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C. Most mutations result from DNA
replication errors.
DNA polymerases sometimes make mistakes that are
not repaired.
DNA damage increases the likelihood of such
mistakes.
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DNA damage can lead to mutation, but is not a
mutation per se because it is not heritable.
...GATCGGATC... ...CTAGCCTAG...
DNA damage (alkylation)
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D. Mutation frequencies are thought to be
roughly similar in all organisms
10-9 to 10-10 / base pair / generation
Thus, in general, mutations are rare.
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E. Mutants can be derived from wild-type
strains (or from other mutant strains).
Wild-type the original strain of an organism
isolated from nature
mutation
Mutant an organism with a genome that carries a
mutation
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Mutations in genes that encode proteins can
affect
protein structure protein function the
phenotype of the organisms
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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
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Overview Effect of mutations on protein
structure
mutation
gene
Translation has two possible outcomes (1) a
change in the amino acid sequence, or (2) no
change.
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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
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1.Silent mutations
...TAC... ...ATG...
DNA
RNA
Polypeptide
No change in the polypeptide
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2. Missense mutations
...TAC... ...ATG...
DNA
RNA
Polypeptide
One amino acid is changed in the polypeptide.
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3. Nonsense mutations (mutation results in a
stop codon)
...TAC... ...ATG...
DNA
RNA
Polypeptide
A truncated polypeptide is made.
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B. Deletions
One or more base pairs are lost
ATGAAAGAG....
ATGGAG....
Possible results a. amino acids or
polypeptides can be lost b. frameshifts can
occur (see below)
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C. Insertions
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)
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D. Frameshift mutations
Insertions or deletions that change the
translational frame
ATGCAAGTTG....
one base pair deletion
Two changes in polypeptides are possible (1)
every amino acid downstream of the mutation is
changed, (2) a truncated (shortened) protein is
produced.
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DNA can have 3 reading frames
A T G C A A G T T G A
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Frameshift mutations change the translational
reading frame.
ATGCAAGTTGA.
one base pair deletion
(Frameshifts occur only if insertion or deletion
is in the reading frame section of a
protein-encoding gene.)
ATCAAGTTGA
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E. Inversions chromosomal segment is inverted
...ATGGAAGAG.... ...TACCTTCTC....
...ATTTCCGAG.... ...TAAAGGCTC....
A number of changes in polypeptides are possible.
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F. Duplications chromosomal segment is duplicated
...ATGGAAGAG.... ...TACCTTCTC....
...ATGGAAGGAAGAG.... ...TACCTTCCTTCTC....
A number of changes in polypeptides are possible.
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III. Effects of mutations on protein
function
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)
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1. No effect (common)
Depending on the protein, up to 80 of the amino
acids may only function as spacers.
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2. Loss of function (common)
Examples
a. A change in an amino acid that
participates directly in catalysis (change
in the active site)
wild type (normal protein)
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2. Loss of function (contd.)
b. A change in an amino acid that causes the
protein to misfold.
wild type protein
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3. Partial loss of function, "leaky" (common)
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
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4. Conditional loss of function (common)
e.g. Temperature-sensitive (heat-
sensitive) mutations.
42C
30C
properly folded
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5. Change of function (rare)
e.g. change in specificity
wild type protein
converts lactose to glucose and galactose
converts maltose to 2 glucose
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6. Restoration of function, reversion ("back
mutation") (rare)
mutant protein
nonfunctional
a second mutation
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a. Same site revertants
i. true revertants
A second mutation restores the original DNA
sequence.
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b. Second site revertants (suppressors)
i. intragenic
A second mutation at a different site within the
same gene restores function.
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Example of an intragenic suppressor
a salt bridge between Lys 121 and Asp 44 is
essential to protein folding.
_

Asp 44
Lys 121
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.
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IV. Effects of mutations on phenotype
Mutations can have many different effects on
phenotype.
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A. Loss of enzyme activity
Examples
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.
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B. Loss of regulatory proteins
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
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V. Mutagens
Substances that increase mutation frequency.
In the lab, mutagens can be used to create
mutations for genetic analysis.
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A. Mutation frequencies
Spontaneous mutations occur with a frequency of
about 10-9 / base pair / generation
(10-3 to 10-4 / gene / generation).
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B. Types of mutagens and the mutations they
cause.
1. Base analogs
Compounds structurally similar to the normal DNA
bases
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Thymine
Bromouracil
O
H
Br
N
N
O
H
Bromouracil will be incorporated into DNA in
place of thymine.
During DNA replication, bromouracil can
mispair with guanine and cause point mutations.
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2. Alkylating agents
Compounds that chemically modify DNA bases via
alkylation
During DNA replication modified bases mispair
causing single base pair change (point)
mutations.
Example dimethyl sulfate
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3. Intercalating agents
Chemicals that insert between DNA base pairs.
DNA bases
H-bonds
backbone
Intercalating agents lead to small deletions and
insertions during DNA replication.
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4. Radiation
Ultraviolet light (UV)
O
O
CH3
CH3
H
H
N
N
N
N
O
O
Thymine dimer two "T"s on the same strand
become covalently bonded.
Thymine dimers lead to various replication errors.
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1. Understand how genotype affects phenotype. 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?
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MCB 3020, Spring 2005
Chapter 10 Microbial Genetics II Genetics
Techniques
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Molecular Genetics II Techniques
I. The isolation of mutants II. The Ames'
test III. General recombination IV.
Complementation
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Some laboratory uses of mutations
1. Mutations can help identify genes
involved in particular biological processes.
What genes are involved in obesity?
2. Mutations can help to determine the
function of specific genes.
Knock genes out, see what happens.
(e.g. metabolic pathway genes,
regulatory genes, transport genes)
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Some advantages of mutant studies with bacteria
Bacteria are haploid.
Bacteria are easily grown in large numbers.
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I. The isolation of mutants
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.
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A. Isolation of histidine biosynthetic
mutants in Escherichia coli
(strains with mutations in his biosynthetic genes)
1. Designate a particular E. coli strain as
the wildtype strain (His phenotype).
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3. Treat culture with mutagen (produces
mutations at random locations).
mutagenized culture
dilute and plate on a rich medium
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4. Select or screen for mutant strains that
require histidine for growth (His-
phenotype).
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Replica plating
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.
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5. Prepare pure cultures from strains that
require histidine for growth.
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.
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In practice, the genotype and phenotype of the
mutant strains is indicated as their differences
from the wildtype strain.
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Naming genes and mutations
(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)
histidinol phosphate aminotransferase
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Auxotrophs
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B. How could you isolate mutants in lactose
catabolism?
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.
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MacConkey lactose indicator medium
Detects acids produced from lactose catabolism.
Lac
lac mutants are unable to produce acid and
hence are white.
Lac-
Between 1/1000 and 1/10,000 colonies will have a
mutation in a particular gene.
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C. Screening and Selection
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2. Selection
Identification of particular mutants by using
conditions that prevent the growth of other
cells.
e.g. selection for antibiotic resistance
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II. The Ames test
A test used to identify mutagens
1. Spread 108 His- cells on minimal plates
(no histidine)
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)
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Possible results
test compound 1
test compound 2
control
is not a mutagen
is a mutagen
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III. General recombination
DNA rearrangements involving crossovers between
homologous DNA sequences.
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A. Cellular uses of recombination
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
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B. Genetic crossovers
DNA exchanges used in recombination
"x" is the crossover site
x
breakage of phosphodiester bonds
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reunion of phosphodiester bonds
For general recombination, crossovers only occur
between between homologous (identical or nearly
identical) DNA segments.
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C. Outcomes of recombination
A
B
C
1. identical sequences
X
A
B
C
single crossover
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A
B
C
2. nearly identical sequences
X
a
B
c
single crossover
a
B
C
recombinant sequences
A
B
c
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3. Two circular DNA molecules
X
single crossover
integration
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4. circular linear
X
X
double crossover
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5. single strand exchange
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D. A model for recombination
alignment of homologous sequences
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unwinding (RecBCD)
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strand exchange
From this point there are two main methods of
resolution.
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1. Inner strand resolution (break and
religate inner strands)
inner
break
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2. Outer strand resolution (break and religate
outer strands)
break
outer strands
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religate
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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
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B. How complementation is observed
1. Start with a bacterium with a recessive
mutation resulting in Trp phenotype or any
other phenotype.
chromosome
Mutation 1
A
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2. Introduce a second DNA molecule into the
bacterium by one of several methods.
mutation 1
A
Restoration of the wildtype (in this case
Trp) phenotype is termed complementation.
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C. Key Point
Complementation indicates that the second DNA
molecule (the plasmid) has a good copy of the
chromosomal gene that is mutated.
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Study objectives 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?
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MCB 3020, Spring 2005 Chapter 10 Microbial
Genetics III DNA transfer
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Life evolves. This leads to diversity.
It is likely that all organisms are related to a
single ancestral cell or group of cells.
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Molecular Genetics III DNA Transfer I. DNA
transfer in prokaryotes A. transformation B.
transduction C. conjugation II. Transposable
elements

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I. DNA transfer in prokaryotes
A. transformation B. transduction C.
conjugation
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DNA transfer in prokaryotes
The transfer of donor DNA to the genome of a
recipient cell.
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Uses of DNA transfer
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.
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A. Transformation
Transfer of free DNA to a bacterial genome.
free DNA
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free DNA
chromosome
recipient cell
general recombination
transformant
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free DNA (plasmid)
transformant
General recombination is not necessary because
plasmids have origins of replication
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1. Competence
The capacity of cells to take up free DNA.
a. Some cells are naturally competent
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Electroporation
cells DNA
electrode
- electrode
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B. Transduction
Transfer of DNA by viral particles
1. Generalized transduction 2. Specialized
transduction
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1. Generalized transduction
In generalized transduction, transducing
particles formed by packaging errors can contain
DNA from any part of the donor genome.
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Generalized transduction
donor cell
chromosome
viral replication
many viruses
a few transducing particles containing part
of donor cell DNA (formed by packaging errors)
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transducing particle
recipient chromosome
recipient cell
general recombination
transductant
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2. Specialized transduction
donor cell (lysogen)
chromosome
prophage (integrated bacterial virus)
improper excision
viral replication
transducing particles
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recipient cell
transducing
virus
chromosome
phage integration
general recombination
transductants
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Key points for specialized transduction
Transducing viruses formed by improper excision
can only transfer DNA adjacent to the prophage
insertion site.
Transducing viruses can become part of the
recipient genome by general recombination or
integration.
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C. Conjugation
Direct cell to cell DNA transfer involving
certain plasmids.
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1. Conjugative plasmids
plasmids that mediate their own transfer
e.g. F-plasmid
IS
tra
IS
oriT
oriS
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2. The DNA transfer process
donor cell (F)
F- pilus
chromosome
F plasmid
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replication and transfer of ssDNA
exconjugant
F
F
Note that the recipient cell becomes F
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3. The F-plasmid can integrate into the
bacterial chromosome.
F plasmid
chromosome
integration
integrated F plasmid
Hfr strain
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Integrated F plasmids can transfer the
chromosome.
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a. Hfr strain
A cell that carries the integrated F plasmid is
called the "Hfr" strain Hfr stands for "high
frequency of recombination"
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Hfr strain
A bacterium with an F plasmid integrated into its
chromosome.
Hfr strain
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).
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b. The integrated F plasmid can mobilize
the chromosome (i.e. move part of the
chromosome to another cell)
Hfr strain
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Bacterial genetic maps
maps that show the relative locations of genes.
X
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c. The integrated F-plasmid can excise
improperly, forming a plasmid with part of
the host chromosome. The resulting plasmid
is called F-prime (F').
Hfr strain
integrated F plasmid
Improper excision
F'
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F-prime (F')
An improperly excised F plasmid containing a
segment of bacterial chromosomal DNA.
Hfr strain
improper excision
F plasmid
F'
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F-prime (F')
segment of bacterial chromosome
part of F plasmid
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III. Transposable elements A. examples 1.
insertion sequences 2. transposons B.
transposition C. uses of transposable elements
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Transposable elements
DNA segments that can move from one location to
another "mobile DNA"
transposable element
IS
IS
host DNA
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transposable element
IS
IS
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.
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A. Examples of transposable elements
1. Insertion sequences
IS2
tnp
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2. Transposons
Tn5
tnp
kan str ble
IS50L
IS50R
host genes
Transposons typically consist of host
gene(s) flanked by insertion sequences.
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B. Transposition
The movement of a transposable element
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Transposition
One element is duplicated. The first copy stays
at the original site. The second copy is found at
another site.
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C. Uses of transposable elements
1. In the lab, transposon mutagenesis
2. In nature, function is not known. Just
selfish DNA?
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Study objectives 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.