On Tap

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High Frequency of Recombination(Hfr)

• ...bacteria exhibiting a high frequency of
  recombination,
• the F factor is integrated into the chromosomal
  genome.

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F factor and Chromosomal DNA are Transferred
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Recombination Requires Crossing over
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Incomplete Transfer of DNA

• Interrupted Mating a break in the pilus during
  conjugation stops the transfer of DNA,
• Transfer occurs at a constant rate,
• provides a means to map bacterial genes.

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How Do You Interrupt Bacterial Mating
spread on agar
mate for specified time
frappe
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Hfr and Mapping
HfrH strs (sensitive to streptomycin) thr
(able to synthesize the amino acid threonine)
azir (resistant to sodium azide) tonr
(resistant to bacteriophage T1) lac (able to
grow with lactose as sole source of carbon) gal
(able to grow with galactose as sole source of
carbon)
F- strr (resistant to streptomycin thr-
(threonine auxotroph) azis (sensitive to sodium
azide) tons (sensitive to phage T1) lac-
(unable to grow on lactose) gal- (unable to
grow on galactose)
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Hfr and Mapping
HfrH strs (sensitive to streptomycin) thr
(able to synthesize the amino acid threonine)
F- strr (resistant to streptomycin) thr-
(threonine auxotroph)
Streptomycin kills the HfrH cells in the mating
mix.
No threonine kills the F- cells in the mating mix.
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Hfr and Mapping
HfrH azir (resistant to sodium azide) tonr
(resistant to bacteriophage T1) lac (able to
grow with lactose as sole source of carbon) gal
(able to grow with galactose as sole source of
carbon)
F- azis (sensitive to sodium azide) tons
(sensitive to phage T1) lac- (unable to grow on
lactose) gal- (unable to grow on galactose)
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Interrupting Bacterial Mating
spread on selective media
mate 9 min
blend
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Replica Plating
After 9 minutes, only azide resistant cells grow.
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10 Minutes
Azide, and bacteriophage resistant cells grow.
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15 Minutes
Azide, and bacteriophage resistant cells, and
lactose utilizing cells.
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18 Minutes
All recombinants grow.
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cells with markers
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Bacterial Map Distances
units minutes
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F factor inserts in different regions of the
bacterial chromosome,
Also inserts in different orientations.
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Replication Origin
Hfr Order of transfer strain
H thr azi ton lac pur gal his gly thi 1
thr thi gly his gal pur lac ton azi
2 lac pur gal his gly thi thr azi ton
3 gal pur lac ton azi thr thi gly his
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F factor
A
a
Hfr
F-
Hfr DNA that is not incorporated in the F-
strand, and DNA that has crossed out of the F-
strand is digested.
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F factor
A transfers first.
A
A
Hfr
F-
A transfers last.
Leading Gene the first gene transferred is
determined empirically.
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Hfr Order of transfer strain
H thr azi ton lac pur gal his gly thi 1
thr thi gly his gal pur lac ton azi
2 lac pur gal his gly thi thr azi ton
3 gal pur lac ton azi thr thi gly his
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Microbes in the news.
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Hfr and Mapping
HfrH strs (sensitive to streptomycin) thr
(able to synthesize the amino acid threonine)
azir (resistant to sodium azide) tonr
(resistant to bacteriophage T1) lac (able to
grow with lactose as sole source of carbon) gal
(able to grow with galactose as sole source of
carbon)
F- strr (resistant to streptomycin thr-
(threonine auxotroph) azis (sensitive to sodium
azide) tons (sensitive to phage T1) lac-
(unable to grow on lactose) gal- (unable to
grow on galactose)
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Hfr and Mapping
HfrH strs (sensitive to streptomycin) thr
(able to synthesize the amino acid threonine)
F- strr (resistant to streptomycin) thr-
(threonine auxotroph)
Streptomycin kills the HfrH cells in the mating
mix.
No threonine kills the F- cells in the mating
mix, also, azide, T1 phage, and a lack of
carbon source.
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Hfr and Mapping
HfrH azir (resistant to sodium azide) tonr
(resistant to bacteriophage T1) lac (able to
grow with lactose as sole source of carbon) gal
(able to grow with galactose as sole source of
carbon)
F- azis (sensitive to sodium azide) tons
(sensitive to phage T1) lac- (unable to grow on
lactose) gal- (unable to grow on galactose)
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Bacterial Map Distances
units minutes
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E. coli Map

• 0 minutes is at the threonine,
• 100 minutes is required to transfer complete
  genome,

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Typical Problem
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combine
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combine
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Refer to partial maps for map distances.
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Practice

• Insights and Solutions, 2,
• Problem 7.17, 7.18, 7.19.

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Transformation

• heritable exchange brought about by the
  incorporation of exogenous DNA,
• usually DNA from same, or similar species.

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Donor and Recipient
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Competence

• a transient state or condition in which a cell
  can bind and internalize exogenous DNA molecules,
• often a result of severe conditions,
• heat/cold,
• starvation, etc.

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Competent Cell
Genes are expressed that produce proteins that,
in turn, span the cell membrane.
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Exogenous DNA Binds Receptor
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Complementary Strand Degraded
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Exogenous DNA Incorporated
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Cell Divides
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Transformation and Mapping

• transformed DNA is generally 10,000 - 20,000 base
  pairs in length,
• carries more than one gene,
• When two or more genes are received from the same
  transformation event, they are said to be
  co-transformed.

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Linkage in Bacteria

• genes that are closer together, have a higher
  probability of being co-transformed,
• higher probability of being on same donor DNA,
• lower chance of crossover event between genes,
• probability of transformation by two separate
  events is low,
• linkage in bacteria refers to proximity.

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Transposable Elements

• a segment of DNA that can move to, or move a
  copy of itself to another locus on the same or a
  different chromosome (hopping DNA),
• may be a single insertion sequence, or a more
  complex structure (transposon) consisting of two
  insertion sequences and one or more intervening
  genes.

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Transposable Elementsmobile DNA
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Why Transposons?

• the DNA sequence between the transposable
  elements may confer an adaptive advantage,
• or at differing dosages,
• upon mobilization, the transposon may hop into
  a part of the genome that is being expressed at a
  higher or lower rate,
• other?

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Recombinases

• Enzymes that catalyze recombination via
  crossing-over event,
• just as sister chromatids can recombine during
  Prophase I,
• any DNA can cross-over and recombine under the
  right circumstances.

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Cre/lox Recombination
The enzyme Cre recombinase associates
specifically with the loxp locus, - the gene
that codes for Cre is elsewhere in the genome,
and is under transcriptional control.
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Integrons
Site specific recombinase, plus adjacent
recognition region
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Integron Excision
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Hop In, Hop Out

• the transposable elements, transposons and
  integrons, etc. may confer a temporary advantage,
  
• once the selective pressure is over, the
  transposable element can re-mobilize and exit a
  disrupted gene, and in many cases return the gene
  to its original state,
• may transpose to a conjugative plasmids, or near
  Hfr integration sites for wide spread dispersal,
• integron cassettes can also excise, and picked
  up by other genetic elements.

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And, Self-Mutate?

• transposable elements are often mobilized during
  environmental stress,
• cassettes are shuttled from cell to cell, etc.
• for example out of billions of cells, one cell
  may have a transposable element that inactivates
  a specific gene,
• upon inactivation, the cell may have an adaptive
  advantage.

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Transposition
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T4 Bacteriophage

• infects E. coli,

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Transduction

• virally mediated gene transfer from one
  bacterium to another,
• bacteria viruses are termed bacteriophages.

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Two Bacteriophage Strategies

• Lytic,
• a type of viral life cycle resulting in the
  release of new phages by death and lysis of the
  host cell,
• Lysogenic,
• a type of viral life cycle in which the visus
  becomes incorporated into the host cells
  chromosome.

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Lytic Cycle
2. host cell physiology is used for phage work,
3. phage DNA replicated, capsule parts made,
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Generalized Transduction

• enzymatic process which can result in the
  transfer of any bacterial gene between related
  strains of bacteria.

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Phage Infects Host
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Phage Hijacks the Host Cells Transcription/Transl
ation Machinery
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Cell Lyses, Phage Move On
C is packaged instead of phage DNA in one of
thousands of new phages,
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End of the Route
Host Chromosome,
Phage DNA,
packaged host DNA,
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Virulent Phages

• reproduce via the lytic cycle only.

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Two Bacteriophage Strategies

• Lytic,
• a type of viral life cycle resulting in the
  release of new phages by death and lysis of the
  host cell,
• Lysogenic,
• a type of viral life cycle in which the visus
  becomes incorporated into the host cells
  chromosome.

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Lytic vs Lysogenic
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Lysogeny

• the integration of viral DNA into the bacterial
  genome,
• a virus that can integrate into the genome is
  termed temperate,
• an integrated phage is termed a prophage.

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Prophage

• non-virulent units that are inserted in the host
  chromosome, and multiply via binary fission along
  with the host DNA,
• prophage can re-enter the lytic cycle to
  complete the virus life cycle.

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Phage Induction

• prophage express a repressor protein that
  inhibits further infection,
• also inhibits prophage DNA excision genes, and
  genes used during the lytic cycle,
• environmental cues (especially events that
  damage DNA) block the expression of the repressor
  protein,
• prophage excises and enters a lytic cycle.

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Specialized Transduction

• upon excision of the prophage, adjacent host DNA
  is taken along,
• the completion of the lytic cycle and subsequent
  infection of another host moves the flanking DNA
  to another bacterium.

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Normal Excision
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Abnormal Excision
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Transfer to Other Cells
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Bacteria are Geniuses

• Cloning identical copies,
• Gene therapy insertion of a healthy, or
  functional gene into a organism lacking a good
  gene,
• Harness Mutation to deal with stress and speed
  evolution,
• Defense develop genes to ward off poisons,
  predators, etc., then share the goods,
• Genetic engineering inserting DNA into another
  organism to do your bidding (Friday),

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Phage Infections
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Phage Phenotypes/Genotypes

• Single Phage Infection uniform plaque
  morphology,
• different phage genotypes can yield different
  phage phenotypes.

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Phage Particles Can Recombine
r small plaque
r- large plaque
h clear plaque
h- turbid plaque
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What is a Gene?

• Bead Theory (lt 1950s),
• The gene was viewed as a fundamental unit of
  structure, indivisible by crossing over.
• The gene was viewed as the fundamental unit of
  change (mutation).
• The gene was viewed as the fundamental unit of
  function (parts of genes were not thought to
  contain function).

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Genetic Fine Structure

• Seymore Benzer
• Demonstrated that a gene can be subdivided into a
  linear array of sites that are mutable and that
  can be recombined.
• Paved the way for the understanding that the
  smallest units of mutation and recombination are
  single nucleotide pairs.

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rII

• A mutant T4 phage was known to produce larger,
  ragged plaques
• this mutation was mapped to two genetic loci on
  the phage DNA molecule, rI and rII,
• rII mutants have an altered host range compared
  with wild-type T4.

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rII Host Range
Permissive E. coli strain B is permissive to rII-
Non-permissive E. coli strain K(l) is
non-permissive to rII-
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rII- mutants
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Infect B with two rII- mutants infect K cells
with resultant phage.
Control rII- parents on K plates.
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Intergenic Recombination
rII1 - rII8
X
rII7
x
rII8
Frequency of recombination indicates map distance.
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Why Stop There?

• Deletion Mapping partial deletions in genes can
  be mapped in just the same way as other
  mutations
• in fact, the site of the deletion can be
  determined by defining which previously mapped
  mutations fail to to recombine into a wild-type
  gene.

Please Study, and master A moment to Think, pp.
269
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Deletion Mapping
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Intervals A1 - A6 or B
Subintervals ...break down interval.
Fine map with reversible mutations in
subinterval.
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What Did We Learn?

• Genes are linear arrays of sub-elements,
• the sub-units are alterable by mutation and able
  to recombine (average 2.3 bases),
• mutations are not produced at all locations in a
  gene,
• and are found at higher frequencies at certain
  locations.

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Coming Up Wednesday Plant Biotechnology
bacteria also have plasmids (T Plasmids) that
they transfer to other organisms, upon
infection, the T plasmid enters the host cell,
becomes incorporated in the host genome, and the
T plasmid genes become expressed, Agrobacterium
tumefaceins transfers genes that force plants to
make strange sugars, that only the Agrobacterium
can digest.
Review Friday