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GENETIC RECOMBINATION IN
PROKARYOTES
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Mechanisms of Genetic Exchange
Bacterial ConjugationMechanism of transferring genetic information from
one bacterium to another, followed by recombinationwith the recipient bacteriums genetic material
Transformation
Uptake of DNA from the surrounding medium andrecombination into the recipient bacteriums genetic
material Transduction
Transfer of genetic material from one bacterium toanother via bacteriophage
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Bacterial Conjugation
Discovered by Lederberg and Tatum (1946)
Two auxotroph strains (one was met bio and the
other thr leu thi)
Culture together on complete medium
Subculture on minimal medium
Some cells were prototrophs (10-7
) and 2-3 independentgene mutations (genetic exchange &recombination)
unlikely to create revertants
One strain had provided genetic material to replace
defective genes in the other
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Lederberg and Tatum: More Work
Transfer is unidirectional
Some strains are always donors, some always
recipients in an exchange
Strains designated F+ (fertility, donor) or F-
(recipient)
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Bernard Davis
Demonstrated usinga U-tube culture that
contact between
donor and recipientcells was necessary
for the transfer of
genetic material Now know transfer
through F pilus
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Bacterial Conjugation
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F factor
Fertility conferred by a factor that could be lost
and regained by a strain (from another F+ strain)
Mobile element
now known to be a plasmid (autonomous genetic element)
100 kbp in size
Encodes 20 genes for genetic transfer (plus others)
Nearly always transferred to recipient cell duringconjugation
Converting recipient to F+
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Hfr Strains and Chromosome
Mapping Nitrogen mustard treatment of F+ strain to
induce mutations (1950, 1953))
Mutant had recombination rate of 10-4 (vs. 10-7)Strains called Hfr for high-frequency
recombination
Unlike normal F+ strains, Hfr strains do not
convert recipient cell to F+
Genes transferred at different rates
Some very commonly, some not at all something hadchanged
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Hfr Strains and Chromosome
Mapping Wollman and Jacobs Interrupted Mating
Technique
Allow mating (conjugation) to proceed for specifiedtime and then transfer to blender
Sheer forces terminate transfer through pilus
Used antibiotic sensitive donor and resistant recipientSome genes always transferred sooner than others
Seemed to be a specific order
Chromosome transferred linearly from a specific start point
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Time Map Times when
individualgenes first
observed to
have been
transferred Time could
vary depending
upon Hfr strain
Order same
Start point
varied
Minutes=mapdistance
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Order of Transfer Same, First
Gene and Direction Varies
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First Prokaryotic Genetic Maps
Map units in minutes, not recombination
frequency
E. coli K12 map approximately 100 minutes total
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Conversion
of F+ to Hfr
F plasmid integrates
into host chromosome
Transfer always begins
from one end of
integrated F
One strand of duplex
peeled off and
transferred through
pilus
Second strand synthesis
and recombination
occurs in recipieint
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Hfr to F
Conversion
Integrated F plasmid can
excise
Often includes portion of
host chromosome
New plasmid called F Cell withF is partially
diploid and called a
merozygote (very useful
for studying genetic
regulation in bacterial
systems)
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Discovery ofrec Genes
Mutants isolated with diminished
recombination ability
recA, recB,recC, and recD genes (at first)
RecA protein involved in strand transfer
reaction, integrating donor strand into recipient
duplex (strand displacement)RecBCD complex cuts and unwinds strand
from donor duplex
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Transformation
Foreign DNA enters the cell from the
surrounding medium (Griffiths experiment)
Two steps
Entry of foreign DNA into cell
Replacement by donor DNA of resident DNA
(but sometimes the donor DNA remainsindependent)
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Transformation Process
CompetenceA physiological state which allows the cell to take up
foreign DNA into the cell
Natural competence requires specific receptors on the cell
surface, energy and transport molecules
dsDNA is taken up, one strand is degraded
Surviving strand integrates into recipient chromosome,
forming heteroduplex
Cotransformation identifies linked genes
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Transformation
Process
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Different Types of Bacteriophages
Lytic- infect the cell andforce the replication of theviruses until the cell lyses(or splits the cell open)
Lysogenic- infect the celland integrates its geneticmaterial into the bacterialDNA, remaining dormantuntil the cell shows signs ofstress, when the phagebecomes active and beginsmaking copies of itself.
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Lysis or Lysogeny
Lysis: Infection by phage produces many
progeny and breaks open (lyses) the host
bacterium
Lysogeny: After infection, the phage DNA
integrates into the host genome and resides there
passively
No progeny
No lysis of the host Can subsequently lyse (lysogeny)
Bacteriophage lambda can do either.
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Lysis Lysogeny
UV Induction
L ti l f b t i h T4
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Lyctic cycle of bacteriophage T4
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Phage Genetics
TRANSDUCTION. There are two forms:Generalized Transduction: bacterial rather thanphage DNA is packaged into a phage head. When
another cell is infected, the bacterial DNA isinjected and in a proportion of cases, may beincorporated into the chromosome by homologousrecombination, replacing the existing genes.
Frequency 105 - 108 per cell. More than one genemay be cotransduced - limit = packaging size =~50kbp = ~1% of bacterial chromosome.
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Phage Genetics
Specialized Transduction: Results from
inaccurate excision of an integrated prophage;
some phage DNA is lost and some bacterial genesare picked up and carried to the next host -
therefore phage are usually defective (non-
infectious) and require replication-competent
helper phage to replicate, depending on whichphage genes are lost.
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Bacteriophage
Viruses with bacterial hosts, phage for short Valuable models for genetic research
T4 life cycle
Phage binds to host cellDNA injected into cell
All host DNA replication, transcription stops
Host chromosome degraded, phage DNA
transcribed/replicated, phage proteins synthesizedPhage particles assembled, host cell lysed to release
progeny
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Bacteriophage T4
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T4Life
Cycle
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Lysogeny
Lysogeny
Lysogenic or temperate phage
Occurs when instead of replicating and lysing hostcell, phage integrates its DNA into host chromosome
prophageNo new phage produced
Integrated phage passed on to cell progeny
Cell and progeny immune to further infection by
similar phage Episome
Genetic element that can either replicateindependently or as part of the bacterial chromosome
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Transduction
Zinder and Lederberg, 1952
Studying Salmonella typhimurium
Recovered prototrophs from culture of two
auxotrophs, but no F plasmid present
U-tube experiment still allowed prototroph production
when two auxotrophs remain separated
Filterable agent involvedDNA transfer by bacteriophage P22
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Transduction
Experiment
Prototrophs
recovered 10-5
Filterable
agent (FA)
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Transduction
Process
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Transduction
Transduction mapping uses gene cotransferfrequency
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Bacteriophage Genetics
Bacteriophage undergo genetic recombination
Genetic maps can be constructed by mixedinfection experiments
Simultaneous infection with two different phage
mutants/strains (Seymour & Benzer, 1950s) h+r x hr+ gives some hr and h+r+ progeny
Two lociintergenic recombination
Recombination frequency= (h+r) + (hr+) / totalplaquesX100
Detection of recombinants at 1 per 106
recombination=map distance between genes
Negative interference
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Intragenic exchange (Fine
structure analysis of gene) Seymour & Benzer - rIIlocus of
bacteriophage T4
recombination= c.o in eukaroytes
Occurs between DNA of individual
bacteriophages during simultaneous
infection of the host bacterium E.coli
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Complementation
Also discovered by Benzer studying rIIlocusof bacteriophage T4
rIImutants can lyseE. coli B but notE. coliK12(l)
Simultaneous infection of K12 with certain pairs ofrIImutants did produce plaques
Individual mutants fell into one of 2 groups
Pairs of mutations that produced plaques were said
to complement each other (differentcomplementation groups)
Smallest unit of complementation called a cistron(equivalent to a gene today-smallest functional
genetic unit)
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Mapping Within a Cistron
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4x103
2 x ----------
8x109
=2x0.5x10-6
=0.000001
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Deletion Mapping
Mutants created with segments of the
chromosome deleted
Mutants that failed to complement a deletionmutant possessed a mutated locus (point) within
the deletion
Preliminary mapping of mutants to a general location
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Deletion Mapping
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Benzers Significance
Combining the results from his studies,
Benzer had defined an abstract unit (the
gene) as a mutational and recombinationalunit that was arranged in a specific order
Now- nucleotides composing of DNA
Experiment conducted before 1960s-Classical examples of genetic
experimentation