IGA 8e

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Multiple Integration Sites
One can map the whole E. coli genome by
using different Hfr strains.
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Work on the Following Problem

• Turn this in next Tuesday (21 Feb 2005)
• You have an F- streptomycin resistant strain of
  E.coli that has mutations requiring the addition
  of arginine, cysteine, methionine, phenylalanine,
  and proline to the medium. You have two different
  prototrophic (but streptomycin sensitive) Hfr
  strains. You get the following data from an
  interrupted mating experiment
• Present a map of these genes and the insertions
  of the F-plasmid in these two Hfr strains.
  Describe the media you used to select the
  recombinants.

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Double Recombination is Necessary
A single recombination between an ENDOGENOTE
and an EXOGENOTE produces a linear chromosome
that cannot be replicated. Incorporation of
genes from the EXOGENOTE requires a double
crossover.
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Double Recombination is Necessary
Recombination can be used to map genes
transferred from the Hfr strain to the F- strain.
Which of the recombinants with two of the three
genes showing the Hfr genotype will be more
common?
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Imprecise Excision of the F factor
The F factor integrated in Hfr strains can
excise. If the excision is precise, it simply
reverses the transition from F to Hfr. If the
excision is not precise, it will generate an F
plasmid, which has genes from the bacterial
chromosome. Bacteria with an F plasmid are
partial diploids or merozygotes.
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Plasmids can move genes between species
Broad host range plasmids can replicate in many
different species. Note that both Gram and
Gram- species can be found as sources for genes
in this plasmid. Resistance (or R) plasmids are
a common type.
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Horizontal Gene Transfer

• The mechanisms for exchange of genetic
  information in bacteria raise the question of how
  often foreign genes are transferred in natural
  bacterial populations.
• The availability of complete genome sequences for
  many bacteria has revealed that substantial
  genetic exchange has taken place.
• This can involve genes related to pathogenicity
  or other interesting phenotypes.
• Collectively, this phenomenon is called
  HORIZONTAL (or LATERAL) GENE TRANSFER.
• Horizontal transfer can involve distantly related
  organisms.
• This can include transfer between eukaryotes and
  prokaryotes as well as distantly related groups
  of prokaryotes.
• The major division in the prokaryotes is between
  BACTERIA and ARCHAEA - transfers can involve
  these groups.

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Horizontal Gene Transfer

• Horizontal gene transfer can be illustrated using
  the likely tree of life.
• This phylogeny is largely based upon rRNA
  (ribosomal RNA) data, with the root placed using
  analyses of duplicate genes.

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Horizontal Gene Transfer

• Now consider analyses of ATPase genes...
• Note the presence of (true) bacteria within the
  archaea - these organisms probably received these
  genes from archaea.

• NOTE - Archaea are bold and Eukaryotes are in
  CAPS.

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Horizontal Gene Transfer

• It is also possible to identify genes introduced
  into lineages using data on the nucleotide
  composition - that was used here

• Can horizontal transfer impact eukaryotes?
• Yes (e.g., organelles) - but the type of transfer
  involving a few genes at a time (which appears
  common in bacteria) may be less common.

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Transformation is another Mechanism that Bacteria
use to Exchange Genes
Bacteria also exchange genes by
transformation. Some bacteria are naturally
competent to take up DNA.
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DNA is the Chemical Agent Responsible for
Transformation

• In 1944, O. Avery, C. MacLeod, and M. McCarty
  provided strong evidence that bacterial
  transformation is mediated by DNA.
• This was based upon the transformation of
  avirulent Streptococcus pneumoniae cells with a
  rough colony morphology to a virulent form with a
  smooth colony morphology by using material from
  heat killed virulent bacteria.
• The observation that this transformation was
  possible had been made more than a decade earlier
  (in 1928) by F. Griffith.
• The differences between avirulent and virulent S.
  pneumoniae cells were heritable, so they
  reflected the presence of a gene for the virulent
  phenotype.
• And the chemicals in the heat killed virulent
  bacteria that are responsible for transformation
  are likely to be the genetic material.

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DNA is the Chemical Agent Responsible for
Transformation

• In 1944, O. Avery, C. MacLeod, and M. McCarty
  provided strong evidence that bacterial
  transformation is mediated by DNA.
• Avery, MacLeod, and McCarty isolated DNA from the
  heat killed virulent bacteria and then destroyed
  specific components of the purified material (in
  separate experiments).
• Proteins were destroyed using proteases.
• RNA was destroyed using RNase.
• DNA was destroyed using DNase.
• Only the DNase treatment destroyed the ability of
  the extract to transform the bacteria.
• Although this may seem pretty definitive, there
  are issues
• It is difficult to purify these enzymes to
  eliminate all other activities (e.g., the DNase
  may contain proteases).

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Phage Represent the Third Mechanism
Bacteria can also exchange genes by the process
of transduction, which involves the exchange of
genes through phage. Phage (also called
bacteriophage) are viruses that infect bacteria.
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Bacteriophage
This shows the DNA phage T4. A small set of
genes are encoded by DNA that is injected into
the host. The phage genome directs the synthesis
of new phage. These phage are released, lysing
the host.
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The Lytic Cycle
The lytic cycle is a general phage life
cycle. The phage infects the cell by injecting
its genome, which directs the synthesis of new
phage. The phage are released by lysis of the
host cell.
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Recombination in phage
Phage can recombine if two different genotypes
infect the same host.
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Genetic Mapping in Phages

• Just as in bacteria and eukaryotes, the isolation
  of mutant phages is possible.
• Typical mutants are temperature sensitive for
  growth or unusual plaque morphology mutants.
• Another class of mutants are host range mutants,
  which determine that bacteria that the phage can
  infect.
• Phage genes can mapped by mixing different
  mutants at a high multiplicity of infection
  (moi).
• This results in the infection of bacteria with
  multiple phages.
• MOI is simply the number of phages added (usually
  measured in pfu/ml) divided by the number of
  bacterial cells (cells/ml).
• Mapping is performed in a manner similar to
  mapping in eukaryotes

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Transduction
Sometimes, segments of the host genome are
transferred by phage. This process is generalized
transduction.
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Transduction

• TRANSDUCTION is the transfer of bacterial genes
  in phage particles.
• When phages lyse cells the bacterial genome is
  usually degraded. Sometimes, fragments of the
  bacterial genome similar in size to the phage
  genome will be packaged into phage particles.
• These phages with bacterial DNA can infect other
  bacteria. A small proportion of the time (less
  than 5-10 of the time) the bacterial DNA is
  integrated into the genome of this host.
• Transduction can be used to map closely linked
  genes.
• If we denote the distance between two genes as d,
  then

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Transduction

• Examine the co-equation

• L is the length of the transducing fragment.
• For phage P1 this is 2 minutes.
• The frequency of co-transduction is a proportion
• Rearranging the equation
• Two important factors are considered by this
  equation.
• The size of the transducing fragment.
• The probability that double cross-overs will
  integrate the genes into the bacterial genome.

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Fine-Structure Mapping by Transduction
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Lysogenic Phages
Some phages (e.g., ? phage) integrate into the
host genome. These are called temperate
phages If an Hfr strain with ? integrated
transfers the ? genome to an uninfected host, the
lytic cycle occurs.
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The Process of Transduction

• There are two potential pathways for bacterial
  genes that are packaged into phage particles
• When these phage with bacterial genes packaged in
  them infect cells, the bacterial DNA can have two
  fates.
• Most of the time, there is ABORTIVE TRANSDUCTION.
• The DNA is neither replicated nor integrated into
  the genome.
• A small proportion of the time COMPLETE
  TRANSDUCTION occurs, involving a double
  cross-over.
• Lyosogenic phages often integrate at specific
  sites in bacterial genomes, a fact that leads to
  a distinct type of transduction.
• Although phage excision is usually precise, some
  of the time adjacent genes are excised with part
  of the phage genome and packaged. This leads to
  SPECIALIZED TRANSDUCTION.

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Specialized Transduction

• Imprecise phage excision can result in the
  packaging of genes near the phage integration
  site.
• These genes will be transduced a large proportion
  of the time, so the phage is said to be a
  specialized transducing phage, since it is
  specialized for the genes it integrates near.
• l is a specialized transducing phage for the gal
  and bio genes

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Physical Mapping and Sequencing

• Physical maps are produced by assembling sets of
  overlapping clones (typically large-insert
  clones).
• The highest resolution physical map is the
  sequence.
• Whole genome shotgun is the most popular approach
  to prokaryotic sequencing. Random segments of the
  genome are sequenced and assembled
  computationally.
• When the sequence is compared to the genetic map
  an excellent correlation is typically observed.

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Putting it all together

• Different methods of mapping can be used at
  different scales.
• These can be assembled into a complete map.
• This was done for E.coli and a few other
  bacteria.
• Sequencing is the most popular approach now.