Bacterial Genetics Introduction and Recombination 1 (ppt)

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

Genetic Manipulation and Bacterial Genetics.

M J Larkin

Genetic Manipulation and Bacterial Genetics.

M J Larkin

http://www.qub.ac.uk/mlpage/courses/level3/page.html


Dr Mike Larkin - CommunicationDr Mike Larkin - Communication

• MBC Room 108 – xt 2288 (Belfast 90972288 Diverted to the

DKB)• DKB Questor Centre Room 316

– 3rd Floor Microbiology Laboratory– xt (9097)4390 or (9097)4388 lab’ or (9033) 5577

office• Email [email protected]• Pigeon hole in Biology & Biochemistry office• ALWAYS LEAVE MESSAGE and HOW TO

CONTACT YOU!

• MBC Room 108 – xt 2288 (Belfast 90972288 Diverted to the

DKB)• DKB Questor Centre Room 316

– 3rd Floor Microbiology Laboratory– xt (9097)4390 or (9097)4388 lab’ or (9033) 5577

office• Email [email protected]• Pigeon hole in Biology & Biochemistry office• ALWAYS LEAVE MESSAGE and HOW TO

CONTACT YOU!


Questor Microbiology and Environmental Genomics Lab’

http://www.qub.ac.uk/mlpage/page1/index.html


Course Theme.Course Theme.

A detailed understanding of the molecular principles that are the basis for the generation of genetic diversity in bacteria.Basic knowledge to enable:The application of genomic technology in microbiology research - assignment

A detailed understanding of the molecular principles that are the basis for the generation of genetic diversity in bacteria.Basic knowledge to enable:The application of genomic technology in microbiology research - assignment

°E.coli systems ML°Bacteria other than E. coli JQ°Pathogens SP

°E.coli systems ML°Bacteria other than E. coli JQ°Pathogens SP


Overview of diversityOverview of diversity

Environmental Influence

Environmental Influence

°DNA Replication°Mutation°Repair

ToC, pH, eHsalinity, growth rate,UV radiation, chemicaldamage

ToC, pH, eHsalinity, growth rate,UV radiation, chemicaldamage

°Recombination°Homologous°Non-homologous°Rearrangements°Deletions

DNATransformationDNATransformation

Phage TransductionPhage Transduction

PlasmidConjugationPlasmidConjugation


E.coli systems and recombination: Determinants of diversity: Overall aims ML

E.coli systems and recombination: Determinants of diversity: Overall aims ML

Nine/Ten lectures with Key topics. Homologous recombination and DNA repairRole of methylation and repair.Role of Plasmids; control of replication, transfer and stability. Illegitimate recombination: transposons and integronsRegulation of DNA transposition.You should:

Have a basic grounding for further reading and other systems covered in the course (e.g pathogens).

Be able to critically read key papers in the area.Critically assess the development of ideas to date.

Nine/Ten lectures with Key topics. Homologous recombination and DNA repairRole of methylation and repair.Role of Plasmids; control of replication, transfer and stability. Illegitimate recombination: transposons and integronsRegulation of DNA transposition.You should:

Have a basic grounding for further reading and other systems covered in the course (e.g pathogens).

Be able to critically read key papers in the area.Critically assess the development of ideas to date.


Homologous RecombinationHomologous Recombination

• Basic mechanisms

• Recombination in conjugation

• The Holliday Model

• Meselson Radding Model

• Recombination in intact replicons

• Enzyme complex involved

• Role of other genes

• Biochemistry of recombination

• Basic mechanisms

• Recombination in conjugation

• The Holliday Model

• Meselson Radding Model

• Recombination in intact replicons

• Enzyme complex involved

• Role of other genes

• Biochemistry of recombination


Basic mechanisms/modelsBasic mechanisms/models

• Breaking and Joining of DNA

– Strand exchange. RECOMBINATION

• Types.

– Homologous / general

– Non-homologous / illegitimate

– Site specific

– Replicative / transposition

• Breaking and Joining of DNA

– Strand exchange. RECOMBINATION

• Types.

– Homologous / general

– Non-homologous / illegitimate

– Site specific

– Replicative / transposition

•HOMOLOGOUS RECOMBINATION•Reciprocal exchange•Gene conversion / segregation•replication not required

•HOMOLOGOUS RECOMBINATION•Reciprocal exchange•Gene conversion / segregation•replication not required

THEORIESChiasmatic Janssens 1909

Darlington 1930Copy Choice Belling 1931

Sturtevant 1949Lederberg 1955

THEORIESChiasmatic Janssens 1909

Darlington 1930Copy Choice Belling 1931

Sturtevant 1949Lederberg 1955


Recombination in bacteriaRecombination in bacteria

• Recombination in Transformation• Invasion by Single stranded DNA

• Recombination in Transduction/ Transfection• Invasion by Double stranded DNA (Mismatch repair)

• Recombination in conjugation• Transfer and invasion by Single stranded DNA

• Recombination between intact Double stranded molecules

• Recombination in Transformation• Invasion by Single stranded DNA

• Recombination in Transduction/ Transfection• Invasion by Double stranded DNA (Mismatch repair)

• Recombination in conjugation• Transfer and invasion by Single stranded DNA

• Recombination between intact Double stranded molecules


Recombination in conjugationRecombination in conjugation

Single Stranded DNA Transferred Evidence.........

e.g.. Mate Hfr WT ( i.e. LacZ+

) X F- lacZ Mutant

Single Stranded DNA Transferred Evidence.........

e.g.. Mate Hfr WT ( i.e. LacZ+

) X F- lacZ Mutant

WT LacZ

Single stranded DNA

Mutated LacZ

mRNA

Defective-galactosidase

mRNA

Functioning-galactosidase

WT - InvasionrecA - NO invasionrecBCD - Invasion

WT - InvasionrecA - NO invasionrecBCD - Invasion


Holliday ModelHolliday Model

3’

3’

5’

5’

AT

CG

3’

3’

5’

5’

AT

CG

3’

3’

5’

5’

T

CG

A

C

A

T

G

BRANCH MIGRATION

C

A

G

T

A

C T

G

Chi -intermediate


Holliday Model contd......Holliday Model contd......

EndonucleaseNicking

Resolve

Resolve

OR

T

GA

C

A

CT

G

C

A

G

T

C

A

G

T


Isomerisation of a 1-strand X-over to produce a 2-strand X-over.

Isomerisation of a 1-strand X-over to produce a 2-strand X-over.

OR

3’end 5’end

OR


What happens to mismatched pairs after

recombination? Gene conversion.What happens to mismatched pairs after

recombination? Gene conversion.DNA ReplicationPost-meiotic segregation

A

C

A

C

A

T

G

C

A

C

Excision

A

T

G

C

RepairA

C

Mismatch repair


Meselson- Radding Model for Bacterial Recombination

Meselson- Radding Model for Bacterial Recombination

1. Single strand nick

2. Dissociation of ssDNA

3. Assimilation into D-Loop

4. D-Loop Digestion

5. Ligation

6. Branch migration

7. Complete Recombination


Recombination between Replicons.Chi- intermediates.

Recombination between Replicons.Chi- intermediates.

1975Valenzuela & Inman (-Phage)Potter and Dressler (ColE1 Plasmid)

1975Valenzuela & Inman (-Phage)Potter and Dressler (ColE1 Plasmid)

ColE1 amplified in E.coliby chloramphenicol.up to 1000 copies.

ColE1 amplified in E.coliby chloramphenicol.up to 1000 copies.

Many recombine by homologousrecombinationMany recombine by homologousrecombination

Extract and examineunder EM - highmagnification

Extract and examineunder EM - highmagnification

Either INTERLOCKING

circles

OR Recombinational

intermediates


Demonstration of Chi- intermediates.Demonstration of Chi- intermediates.

Cut ColE1 at single EcoR1 site.Cut ColE1 at single EcoR1 site.

Agarose gel and Et Br stainAgarose gel and Et Br stain

A BAll LinearDNA ?

OR

Some stillJoined ?

Observe using EMObserve using EMEcoR1 Cuts

TWISTChi


Resolution of Chi-intermediates.Resolution of Chi-intermediates.

Role of EndonucleasesRole of Endonucleases

1

2

3

4

Cuts 2 and 4 Cuts 1 and 3 Cuts 1 and 2or 3 and 4


Development of Recombination ModelsDevelopment of Recombination Models

“The prudent Scientist keeps his (sic) hypothesis simple. He endows it with only enough complexity to account for his observations. He stands stubbornly by his simple observations; resisting the complicating results of other people’s experiments, until there is no further doubt of their validity. Then he retreats a very short way, taking up a new position; with only enough added complexity to accommodate the unwanted findings. There he digs in and prepares for the next attack of

the anarchist.” D. Stadler (1973) Annual Review of Genetics Vol7.

“The prudent Scientist keeps his (sic) hypothesis simple. He endows it with only enough complexity to account for his observations. He stands stubbornly by his simple observations; resisting the complicating results of other people’s experiments, until there is no further doubt of their validity. Then he retreats a very short way, taking up a new position; with only enough added complexity to accommodate the unwanted findings. There he digs in and prepares for the next attack of

the anarchist.” D. Stadler (1973) Annual Review of Genetics Vol7.


The question remains the same!The question remains the same!

What is the role of homologous recombination in the evolution of microbial genomes?

1973 – a detailed answer possible – but mechanisms not known and significance of non- homologous recombination uncertain

2006 – mechanisms better resolved – must now discuss in relation to transposons and other mechanisms. Whole genomes data confuse the picture!

What is the role of homologous recombination in the evolution of microbial genomes?

1973 – a detailed answer possible – but mechanisms not known and significance of non- homologous recombination uncertain

2006 – mechanisms better resolved – must now discuss in relation to transposons and other mechanisms. Whole genomes data confuse the picture!

Documents

Bacterial Genetics Introduction and Recombination 1 (ppt)