Regulation at transcriptional level

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14 Δεκ 2012 (πριν από 4 χρόνια και 4 μήνες)

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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

Prokaryotic microbes
: bacteria

Prokaryotic genome


Chromosomal

DNA: double
-
stranded; circular;
haploid
.


Extrachromosomal

genetic
elements


Plasmids

(autonomously self
-



replicating)



Phages

(bacterial viruses)


Transposons

(DNA sequences
that move within the same or
between two DNA molecules)

Eukaryotic microbes
:


fungi, yeasts


Eukaryotic genome


Chromosomal DNA


Mitochondrial DNA


Plasmids in yeasts

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

Tryptophan operon

Regulation at transcriptional level
(Example II)

Negative control


Repressor


Corepressor


Operator

Attenuation

Transcription
termination signal

Mutation

Types of mutations

1. Base substitutions



Silent vs. neutral; missense vs. nonsense

2. Deletions

3. Insertions

4. Rearrangements: duplication, inversion, transposition

may cause
frameshift

or

null
mutation

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

Nucleotide
excision
repair

Base 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)

Icosahedral
tailess

Icosahedral
tailed

Filamentous

Structure and genetic materials of phages


Coat (Capsid)


Nucleic acid

Lysogenic phase

Lytic phase

Life cycle

Phage
l

as an example



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.

Natural transformation

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

Hfr strain

F plasmid

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

F plasmid

--
an episome

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.

Transposons

Mobile genetic elements

May carry drug resistance genes

Sometimes insert into genes and inactivate them
(insertional mutation)

E


Conjugational transposon

Trans
-
Gram
gene transfer

Spread of transposon
throughout a bacterial
population

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