Download rII

High Frequency of Recombination
(Hfr)
...bacteria exhibiting a high frequency of
recombination,
…the F factor is integrated into the
chromosomal genome.
F factor and Chromosomal DNA
are Transferred
Recombination Requires
Crossing over
Double Crossover
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.
How Do You Interrupt Bacterial
Mating
spread on agar
mate for
specified
time
frappe
Hfr and Mapping
HfrH
strs
thr+
azir
tonr
lac+
gal+
(sensitive to streptomycin)
(able to synthesize the amino acid threonine)
(resistant to sodium azide)
(resistant to bacteriophage T1)
(able to grow with lactose as sole source of carbon)
(able to grow with galactose as sole source of carbon)
Fstrr
thrazis
tons
lacgal-
(resistant to streptomycin
(threonine auxotroph)
(sensitive to sodium azide)
(sensitive to phage T1)
(unable to grow on lactose)
(unable to grow on galactose)
Hfr and Mapping
HfrH
strs
thr+
(sensitive to streptomycin)
(able to synthesize the amino acid threonine)
Fstrr
thr-
(resistant to streptomycin)
(threonine auxotroph)
Streptomycin kills the HfrH cells in the mating mix.
No threonine kills the F- cells in the mating mix.
Hfr and Mapping
HfrH
azir
tonr
lac+
gal+
(resistant to sodium azide)
(resistant to bacteriophage T1)
(able to grow with lactose as sole source of carbon)
(able to grow with galactose as sole source of carbon)
Fazis
tons
lacgal-
(sensitive to sodium azide)
(sensitive to phage T1)
(unable to grow on lactose)
(unable to grow on galactose)
Interrupting Bacterial Mating
spread on selective media
mate 9 min
blend
Replica Plating
After 9 minutes, only azide resistant cells grow.
10 Minutes
Azide, and bacteriophage resistant cells grow.
15 Minutes
Azide, and bacteriophage resistant cells, and lactose utilizing cells.
18 Minutes
All recombinants grow.
Bacterial Map Distances
units = minutes
HfrH
F factor inserts in different regions of the bacterial
chromosome,
Also inserts in different orientations.
Replication Origin
Hfr
strain
H
1
2
3
Order of transfer
thr azi ton lac pur gal his gly thi
thr thi gly his gal pur lac ton azi
lac pur gal his gly thi thr azi ton
gal pur lac ton azi thr thi gly his
Indicates direction of transfer.
F factor
Aa
A
aA
Hfr
F-
Hfr DNA that is not incorporated in
the F- strand, and DNA that has
crossed out of the F- strand is
digested.
F factor
A transfers first.
A
A
Hfr
F-
A transfers last.
A
Hfr
A
F-
Leading Gene: the first gene transferred is determined empirically.
Hfr
strain
H
1
2
3
Order of transfer
thr azi ton lac pur gal his gly thi
thr thi gly his gal pur lac ton azi
lac pur gal his gly thi thr azi ton
gal pur lac ton azi thr thi gly his
Microbes…
…in the news.
Hfr and Mapping
HfrH
strs
thr+
azir
tonr
lac+
gal+
(sensitive to streptomycin)
(able to synthesize the amino acid threonine)
(resistant to sodium azide)
(resistant to bacteriophage T1)
(able to grow with lactose as sole source of carbon)
(able to grow with galactose as sole source of carbon)
Fstrr
thrazis
tons
lacgal-
(resistant to streptomycin
(threonine auxotroph)
(sensitive to sodium azide)
(sensitive to phage T1)
(unable to grow on lactose)
(unable to grow on galactose)
Hfr and Mapping
HfrH
strs
thr+
(sensitive to streptomycin)
(able to synthesize the amino acid threonine)
Fstrr
thr-
(resistant to streptomycin)
(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.
Hfr and Mapping
HfrH
azir
tonr
lac+
gal+
(resistant to sodium azide)
(resistant to bacteriophage T1)
(able to grow with lactose as sole source of carbon)
(able to grow with galactose as sole source of carbon)
Fazis
tons
lacgal-
(sensitive to sodium azide)
(sensitive to phage T1)
(unable to grow on lactose)
(unable to grow on galactose)
Bacterial Map Distances
units = minutes
E. coli Map
• 0 minutes is at the
threonine,
• 100 minutes is
required to transfer
complete genome,
Typical Problem
combine
combine
+
Refer to partial maps
for map distances.
11.5 minutes
Join Maps
26 minutes
Practice
• Insights and Solutions, #2,
• Problem 7.17, 7.18, 7.19.
Transformation
• heritable exchange brought about by the
incorporation of exogenous DNA,
– usually DNA from same, or similar species.
Donor and Recipient
Not all cells are competent to receive DNA.
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.
Competent Cell
Genes are expressed that produce proteins that, in turn, span the cell membrane.
Exogenous DNA Binds Receptor
Complementary Strand Degraded
...one strand of the exogenous DNA is degraded also.
Exogenous DNA Incorporated
Heteroduplex
Cell Divides
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.
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.
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.
Transposable Elements
mobile DNA
Transposon: carries one or more genes.
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?
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.
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.
Integrons
Site specific recombinase, plus adjacent recognition region
Integron Excision
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.
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.
Transposition
normal gene, normal
RNA, normal protein,
transposon inserted in
gene, abnormal RNA,
abnormal protein, loss of
function.
T4 Bacteriophage
…infects E. coli,
Transduction
…virally mediated gene transfer from one
bacterium to another,
…bacteria viruses are termed bacteriophages.
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 cell’s
chromosome.
Lytic Cycle
specific transmembrane
phage/bacteria binding
sites,
4. phage reassemble
with repackaged DNA,
1. host cell physiology
is shut down,
virus DNA
inserted into
host cell,
2. host cell physiology
is used for phage work,
3. phage DNA replicated,
capsule parts made,
5. host cell is
degraded and
lyses.
Generalized Transduction
…enzymatic process which can result in the
transfer of any bacterial gene between
related strains of bacteria.
Phage Infects Host
we’ll follow gene C+.
Specific Binding Sites,
Phage DNA inserted,
Upon infection, host cell physiology is shut down,
Phage Hijacks the Host Cell’s
Transcription/Translation
Machinery
gene C+ is present
on a DNA fragment.
Phage replicates own DNA,
makes protein head etc.,
Host cell degraded, the host
chromosome is cut,
Cell Lyses, Phage Move On
C+ is packaged
instead of
phage DNA in
one of
thousands of
new phages,
phage particle
with C+ moves
to another host
cell.
End of the Route
Host Chromosome,
Phage DNA,
packaged
host DNA,
inserted in cell,
inserted in Genome,
via double crossover.
packaged
host DNA,
Virulent Phages
…reproduce via the lytic cycle only.
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 cell’s
chromosome.
Lytic vs Lysogenic
viral DNA is
incorporated into
the host genome.
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.
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.
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.
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.
Normal Excision
Abnormal Excision
flanking DNA is removed.
Transfer to Other Cells
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),
Phage Infections
Phage Phenotypes/Genotypes
Single Phage Infection; uniform plaque morphology,
• different phage genotypes can yield different
phage phenotypes.
Phage Particles Can Recombine
r+: small plaque
r-: large plaque
h+: clear plaque
h-: turbid plaque
What is a Gene?
• Bead Theory (< 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).
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.
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.
rII Host Range
Permissive: E. coli strain B is permissive to rIINon-permissive: E. coli strain K(l) is non-permissive to rII-
rIImutants
Infect B with two
rII- mutants…
…infect K cells
with resultant
phage.
Control: rII- parents on K plates.
Intergenic Recombination
rII1 rII7 rII4 rII5 rII2 rII8 rII3 rII6
rII1 - rII8
rII7
rII8
X
x
Co-transduce on B.
X
Select for wt recombinants on K.
Frequency of recombination indicates map distance.
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
Deletion Mapping
Intervals
A1 - A6 or B
Subintervals
...break down
interval.
Fine map with
“reversible”
mutations in
subinterval.
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.
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