Genetic Manipulation and Bacterial Genetics' M J Larkin

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Genetic Manipulation and Bacterial Genetics.M J
Larkin
http//www.qub.ac.uk/mlpage/courses/level3/page.ht
ml
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Dr 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 m.larkin_at_qub.ac.uk
• Pigeon hole in Biology Biochemistry office
• ALWAYS LEAVE MESSAGE and HOW TO CONTACT YOU!

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Questor Microbiology and Environmental Genomics
Lab
http//www.qub.ac.uk/mlpage/page1/index.html
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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

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

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Overview of diversity
Environmental Influence

• Recombination
• Homologous
• Non-homologous
• Rearrangements
• Deletions

• DNA Replication
• Mutation
• Repair

ToC, pH, eH salinity, growth rate, UV radiation,
chemical damage
DNA Transformation
Plasmid Conjugation
Phage Transduction
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E.coli systems and recombination Determinants of
diversity Overall aims ML

• Nine/Ten lectures with Key topics.
• Homologous recombination and DNA repair
• Role of methylation and repair.
• Role of Plasmids control of replication,
  transfer and stability.
• Illegitimate recombination transposons and
  integrons
• Regulation 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.

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Homologous 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

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Basic mechanisms/models

• 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

THEORIES Chiasmatic Janssens 1909 Darlington
1930 Copy Choice Belling 1931 Sturtevant
1949 Lederberg 1955
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Recombination 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

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Recombination in conjugation
Single Stranded DNA Transferred
Evidence......... e.g.. Mate Hfr WT ( i.e.
LacZ) X F- lacZ Mutant
mRNA
Defective ?-galactosidase
mRNA
WT - Invasion recA - NO invasion recBCD - Invasion
Functioning ?-galactosidase
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Holliday Model
BRANCH MIGRATION
Chi -intermediate
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Holliday Model contd......
OR
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Isomerisation of a 1-strand X-over to produce a
2-strand X-over.
OR
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What happens to mismatched pairs after
recombination? Gene conversion.
DNA Replication Post-meiotic segregation
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Meselson- Radding Model for Bacterial
Recombination
1. Single strand nick
5. Ligation
2. Dissociation of ssDNA
6. Branch migration
3. Assimilation into D-Loop
7. Complete Recombination
4. D-Loop Digestion
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Recombination between Replicons.Chi-
intermediates.
1975 Valenzuela Inman (?-Phage) Potter and
Dressler (ColE1 Plasmid)
Extract and examine under EM - high magnification
ColE1 amplified in E.coli by chloramphenicol. up
to 1000 copies.
Many recombine by homologous recombination
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Demonstration of Chi- intermediates.
Cut ColE1 at single EcoR1 site.
Agarose gel and Et Br stain
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Resolution of Chi-intermediates.
Role of Endonucleases
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Development 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
  peoples
• 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.

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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!