prokaryotic genetics

8/4/2019 Prokaryotic Genetics

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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

prokaryotic genetics

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