Transcript Replication of chromosomal DNA

Microbial Genetics
Genomic structure
Replication of chromosomal DNA
Regulation of gene expression
Mutation, repair and recombination
Gene exchange in bacteria
Genetic engineering
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何漣漪老師
Genomic structure
Eukaryotic microbes:
fungi, yeasts
Eukaryotic genome
Chromosomal DNA
Mitochondrial DNA
Plasmids in yeasts
Prokaryotic microbes: bacteria
Prokaryotic genome
Chromosomal DNA: doublestranded; circular; haploid.
Extrachromosomal genetic
elements
Plasmids (autonomously selfreplicating)
Phages (bacterial viruses)
Transposons (DNA sequences
that move within the same or
between two DNA molecules)
Replication of chromosomal DNA
Replication of
bacterial genome
requires:
Replication origin
(oriC)
DNA polymerase
Primase
Helicase
Topoisomerase
Semiconservative
Bidirectional
Control of gene expression
A bacterial cell may regulate the expression of a
certain set of its own genes in response to an
environmental change by a variety of mechanisms.
Alternative s factors (e.g. sporulation)
Regulators (activators or repressors) activated
or inactivated by an inducer or corepressor (e.g.,
cAMP in utilization of lactose; auto-inducers in
quorum-sensing)
Attenuation
Regulation at transcriptional level
(Example I)
Operon
Negative
control
Repressor
Inducer
Operator
Lactose operon
Positive
control
Activator
Inducer
Lactose operon
Regulation at transcriptional level
(Example II)
Negative control
Repressor
Corepressor
Operator
Tryptophan operon
Attenuation
Transcription
termination signal
Types of mutations
Mutation
1. Base substitutions
Silent vs. neutral; missense vs. nonsense
2. Deletions
may cause frameshift or null mutation
3. Insertions
4. Rearrangements: duplication, inversion, transposition
Spontaneous
mutations
Cuased by tautomeric
shift of the nucleotides
which lead to replication
errors
Induced mutations
Physical mutagens:
e.g., UV irradiation
(heat, ionizing radiation)
Chemical mutagens
Base analog
Frameshift
intercalating agents
Base modification
Transposable elements
DNA Repair
1. Direct DNA repair
(e.g., photoreactivation)
2. Excision repair
Base excision repair
Nucleotide excision repair
3. Mismatch repair
4. SOS response
5. Error-prone repair
Thymine-thymine dimer
formed by UV radiation
Excision
repair
Base excision
repair
Nucleotide
excision
repair
Base excision
repair
Nucleotide
excision
repair
SOS repair in bacteria
1. Inducible system used only when error-free
mechanisms of repair cannot cope with
damage
2. Insert random nucleotides in place of the
damaged ones
3. Error-prone
Gene exchange in bacteria
Mediated by plasmids and phages
Plasmid
Extrachromosomal
Autonomously replicating
Circular or linear (rarely)
May encode drug resistance
or toxins
Various copy numbers
Some are self-transmissible
Bacteriophage (bacterial viruses)
Structure and genetic materials of phages
Coat (Capsid)
Nucleic acid
Icosahedral
tailess
Icosahedral
tailed
Filamentous
Life cycle
Phage l as an example
Lytic phase
Lysogenic phase
Virulent phages: undergo
only lytic cycle
Temperate phages:
undergo both lytic and
lysogenic cycles
Plaques: a hollow formed
on a bacterial lawn
resulting from infection of
the bacterial cells by
phages.
Mechanisms of gene transfer
Transformation: uptake of naked exogenous DNA by
living cells.
Conjugation: mediated by self-transmissible plasmids.
Transduction: phage-mediated genetic recombination.
Transformation
Natural transformation
Artificial transformation
(conventional method
and electroporation)
Demonstration
of
transformation
Avery, MacLeod, and
McCarty (1944)
Conjugation
mediated by
self-transmissible plasmids
(e.g., F plasmid; R plasmids)
F plasmid
F plasmid
--an episome
F plasmid can integrate into
bacterial chromosome to
generate Hfr (high frequency
of recombination) donors
Excision of F plasmid can
produce a recombinant F
plasmid (F’) which contains
a fragment of bacterial
chromosomal DNA
Hfr strain
F’ plasmid
Transduction
phage-mediated genetic recombination
Generalized v.s. specialized transduction
Mechanism of Recombination
Homologous recombination
Site-specific recombination
Transposition
Illegitimate recombination
Intermolecular
Intramolecular
Double
crossover
Homologous recombination
Importance of gene transfer to bacteria
• Gene transfer provide a source of genetic
variation in addition to mutation that alters
the genotype of bacteria. The new genetic
information acquired allows the bacteria to
adapt to changing environmental conditions
through the process of natural selection.
Drug resistance (R plasmids)
Pathogenicity (bacterial virulence)
• Transposons greatly expand the opportunity
for gene movement.
Mobile genetic elements
Transposons
May carry drug resistance genes
Sometimes insert into genes and inactivate them
(insertional mutation)
E
Conjugational transposon
Spread of transposon
throughout a bacterial
population
Trans-Gram
gene transfer
Cloning
Cloning vectors
plasmids
phages
Restriction enzymes
Ligase
In vitro phage packaging
Library
construction
Genomic library
cDNA library
Applications of genetic engineering
Construction of industrially important bacteria
Genetic engineering of plants and animals (transgenic
pants or animals)
Production of useful proteins (e.g. insulin, interferon, etc.)
in bacteria, yeasts, insect and mammalian cells
Recombinant protein (e.g. HBsAg) vaccines and DNA
vaccines
Double-strand
break repair
(postreplication
repair)
End-joining
(error-prone)
Translocation
Short deletion at
the joining point