Upload
angel-butler
View
219
Download
3
Embed Size (px)
Citation preview
©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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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!
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Questor Microbiology and Environmental Genomics Lab’
http://www.qub.ac.uk/mlpage/page1/index.html
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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.
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Holliday Model contd......Holliday Model contd......
EndonucleaseNicking
Resolve
Resolve
OR
T
GA
C
A
CT
G
C
A
G
T
C
A
G
T
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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.
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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!