Genetic Exchange in Bacteria

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Genetic Exchange in Bacteria

• Transformation
• Conjugation
• Transduction

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Fate of Foreign DNA in Bacteria

• Restriction and modification
• Recombination
• Replication

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Restriction-Modification Systems

• Discovery
• Mechanism
• Types
• As a powerful tool

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Discovery of Restriction-Modification Systems

• Discovered in 1960s and 1970s by Werner Arber,
  Hamilton O. Smith, and Daniel Nathans. Nobel
  Laureates in 1978.

Experiment materials Three different E. coli
strains E. coli B, E. coli K and E. coli
C. A phage. EOP Efficiency of
plating Number of plaques successfully formed
divided by total phages used.
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(No Transcript)
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Explanation a) When phages first infected
bacteria, only a small portion (0.01) of
phages survived. (restriction) b) Survived
phages were modified in such that all
phages survived in second round.
(modification). c) Two E. coli strains have
different R-M systems.
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E. coli K and E. coli B have different
restriction-modification systems. E. coli C
does not have a restriction system.
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Immigration Control Region Or Immigration Island
percentage of GC
(host specificity for DNA)
hsdS specificity subunit that recognizes the DNA
sequence For E. coli K
AACNNNNNNGTGC For E. coli B
TGANNNNNNNNTGTC
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hsdR produces a restriction subunit that cleaves
the unmethylated DNA. hsdM produces a
methyltransferase to methylate DNA. mcrBC
restricts DNA containing methyl cytosine
residues. (specifically modified DNA) mrr
restricts DNA with m6-methyl adenine or m6-methyl
cytosine. (specifically modified DNA)
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Antirestriction Systems

• Loss of restriction sites.
• If DNA does not have the restriction sites, it
  will not be cleaved.
• Modified bases
• Hydroxylmethylcytosine instead of cytosine.

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Antirestriction Systems

• Self-methylation
• Specific methylase to modify their DNA.
• Activation of a host methylase
• To methylate DNA before being cleaved.

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Antirestriction Systems

• Degradation of host S-adensylmethione
• S-adensylmethionine hydrolase
• Inhibit host restriction endonuclease
• Antirestriction proteins (Ard) inhibits type I
  enzyme
• A motif that is very similar to a motif found in
  the HsdS subunit.
• DNA repair systems
• Repair the cleaved DNA for surviving

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Restriction-Modification Systems

• Type I recognize a non-symmetric specific
  sequence, but cleave randomly (out of recognition
  site). (usually coded by bacteria chromosome)
• Type II recognize a symmetric sequence, usually
  cut within the site.
• Type III recognize a symmetric sequence, but
  cleaves outside of the recognition site
  (intermediate between Type I and Type II).

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Type I Restriction Enzymes

• Contain multiple subunits
• a specificity subunit S which is responsible
  for DNA sequence recognition (HsdS)
• a methylation subunit M which methylates DNA
  (HsdM).
• a restriction subunit R which cleaves DNA at an
  unmethylated restriction site. (HsdR).
• Require ATP, Mg2, and S-adenosyl-methionine
  (Sam).

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Type III Restriction Enzymes

• Contain multi-subunits
• recognize specific sequences, but typically
  cleave 5 to 25 nucleotides away.
• the restriction activity requires ATP and is
  enhanced by S-adenosyl-methionine.
• Only five such enzymes have been characterized so
  far.
• Not very useful in manipulation of DNA.

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Type II Restriction Enzymes

• Contain two subunits
• One subunit acts as a restriction enzyme (HsdR)
• The other subunit acts as a methylase (HsdM).
• the restriction enzyme requires only Mg2 as a
  cofactor.
• Most of the known enzymes recognize symmetric
  sequences and cleaves symmetrically.
• 5GAATTC 3-
• 3CTTAAG 5-
• There are more than 2,500 enzymes that recognize
  over 200 specific sites.

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palindromic sequence
5' - A A T T C - 3 3'-G - 5'
5' - G A A T T C - 3 3' - C T T A A G - 5'
5' - G 3' - C T T A A- 5'
EcoR 1
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Four-hitters, Six-hitters and Eight-hitters
Four-hitters e.g. Aci I 5' CCGC 3
3GGCG5'
Six-hitters e.g. EcoR I 5' GAATTC 3
3CTTAAG5'
Eight-hitters e.g. Pac I 5' TTAATTAA 3
3AATTAATT 5'
Some exceptions.
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Blunt ends and Cohesive ends
Blunt ends e.g. Sma I. 5-CCCGGG-3
3-GGGCCC-5 Cohesive ends e.g. Xma I
5-CCCGGG-3 5 cohesive end
3-GGGCCC-5 Xma I is an isoschizomer of SmaI.
Kpn I 5-GGTACC-3 3 cohesive
end 3-CCATGG-5
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Physical Map
Physical map of DNA shows the actual distance
between Different positions in the molecule.
Hind III
EcoR1
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Physical Map
Hind III
EcoR1
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Electrophoresis