phage bacteria

8/13/2019 Phage Bacteria

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Genetics of bacteria and phages


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Bacteria and phages in genetic research:

Advantages: They are haploid, therefore recessiveness or dominance dont come into

play except in the case of partial diploids. New generations are produced within one or

two days. They are easy to grow in enormous numbers, this allows for analysis of rare

genetic events.The offspring is clonal and genetically identical.

Recombination is quite different in prokaryotes:

A bacterial cell contains one circular chromosome and rarely encounters another

complete chromosome. Recombination is usually between a chromosomal fragment

from a donor cell and an intact chromosome of the recipient cell.Incorporation of part of the transferred donor DNA requires at least two or any even

number of exchange events because the recipient DNA is circular. Furthermore,

recombination is not reciprocal and produces only one recombinant molecule, the

circular recipient molecule, containing an integrated piece of donor DNA.

A single bacterial cell placed on solid medium will grow exponentially and form a clonallyderived colony. The appearance of colonies and the growth requirements of bacteria

can be used to identify the genotype of the colonies.


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Genetic analysis of bacteria by mutation analysis:

Different types of mutations can be used:

Antibiotic resistance: these mutants are able to grow in the presence of an antibiotic,

such as streptomycin.

Nutritional mutants: Wild-type bacteria can synthesize most of the complex nutrients

they require from simple molecules present in the minimal growth medium and arehence called prototrophs.

This ability to grow on simple or minimal media can be lost due to mutation of enzymes

involved in the synthesis of nutrients. Mutants unable to synthesize an essential nutrient

cannot grow unless that nutrient is supplied in the growth medium, and are called

auxotrophs.

Carbon-sourcemutants cannot utilize particular substances as energy sources, suchas lactose and are unable to form colonies on medium containing lactose as the sole

energy source.

A medium in which wild-type cells form colonies is called nonselective medium. Mutant

cells an wild-type cells are not distinguishable on nonselective medium. If the medium

allows only one type of cell to grow it is termed selective. Eg. Medium containingstreptomycin is selective for Strp-r (streptomycin resistant) phenotyptes.

In bacteria phenotypes are designated with 3 letters (first letter capitalized) , a

superscript denoting absence or presence of a character and s and r denoting

resistance or sensitivity. For example Strp-r or Leu-,a genotype is lowercase italics: leu-.


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Recombination

process

Cell contact

required?Sensitive to DNase?

Criterion

Transformation no yes

Conjugation yes no

Transduction no no


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U-tube experiment:

Bacteria of different genotypes are

in different arms of the tube,

separated by sintered glass filter

that prevents cell-cell contact.

This experiment tests the transferof genetic material. DNase in the

culture medium degrades free

DNA, providing a test for

transformation. If recombination

occurs, it is likely taking place by

means of transduction.Why transduction? Bacteria are

too large to make it through the

filter, that excludes conjugation.

Transformation is excluded

because the DNA in the media

would get degraded by DNA. In aphage, however, the DNA is

packaged in protein and protected

from digestion.


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Transformationinvolves the uptake of DNA from donor cells and

integration into the host recipient genome. In nature DNA becomes

available from breakage of donor cells. Transformation efficiencies can be

increased significantly by chemical treatment that makes the cells

competent. The uptake efficiency is rather poor, even if cells are madecompetent it is 1 in 1000 cells.

Transformation can be detected by selection for inheritance of a phenotype

from the donor DNA by the recipient cells. For example, purified DNA from

an erythromycin resistant strain of S. pneumoniae (Eryr) is mixed with cells

from a culture of a sensitive strain (erys). After a period of incubation (time

for DNA uptake and expression by the donor) cells are plated on

erythromycin. The formation of eryrcolonies is significantly above the

mutation rate, this indicates that transformation has occurred.

Transformation can be useful for gene mapping. If two genes are

separated far enough that most of the times the DNA fragment is broken

before transformation, then the probabilities of cotransformation is the

same as the product of probability for the individual genes i.e. 103x103. If

they are close enough the frequency of cotransformation is substantially

greater


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Transformation:

Griffith discovered transformation in 1928, 17 years later Avery, MacLeod and

McCarty demonstrated that DNA was the transforming principle.

What is itsimportancebesides these discoveries?

It is important in genetic analysis of yeast, and bacteria, and it is central to most

cloning strategies. Although not necessarily the most efficient, it is the

simplest form of DNA transfer.

Griffiths discoveries could be made in Pneumococcus because these cells can

become naturally competent to take up DNA. Natural competencefor

transformation is observed in Bacillus subtilis, Haemophilus influenzae, Str.

Pneumoniae etc. and people thought for some time it is limited to thesespecies, but not so. It contributes to antigenic variation in gonococcus

(Neisseria gonorrhoeae) where it involves pil genes, which are also involve in

attachment to epithelial cells.

General features: Competence occurs usually in the late log phase of growth,

possibly as a response to increasing cell density, nutrient depletion andaccumulation of secreted competence factors. (Natural competence may only

last for a short time period). These factors stimulate gene expression of other

genes required for competence through a 2 component regulatory system

(transmembrane signal transduction). It could be considered a form of quorum

sensing. In B. subtilis some genes involved in transformation also areimportant in early sporulation.


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In naturally competent S.pneumoniae (gram-positive) cells, double-stranded

DNA fragments bind to cell surface receptors, however, only one strand is

taken up. In some species this can be specific because it depends on the

presence of species-specific DNA on the fragments. Eg. In Neisseria

menengitidis DNA uptake is dependent on a 10bp sequence, of which there are2000 copies through out the N, menengitidis genome. H. influenzae

transformation requires a 29bp sequence which occurs 1500 times in the

genome. In other species, such as B. subtilis, and Str. pneumoniae the uptake

is non-specific and occurs with almost any linear piece of DNA.

H.influenzae takes up double stranded DNA.

However, transformation requires integration into the host genomein the

case of linear (non-plasmid) sequences and is facilitated by sequence

homology. Therefore, the more similar the sequences are to the host genome,

the more likely is a recombination event with with the host genome. That is, the

recombinant events lead to substitution rather than addition of DNA sequences.

Str. Pneumoniae has apparently become penicillin resistant as a result of

penicillin target gene replacement with the resistant gene from resistant

streptococci.


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Following uptake of DNA, the transforming donor DNA replaces or

substitutes the recipient DNA through homologous recombination events

involving RecA like proteins. DNA is not just added or inserted into the host

genome.


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Mapping by transformation:

When transformation is used to transfer genes to measure recombination

frequencies between markers several difficulties arise. The probability of

recovering the donor marker in the recipient depends on the molecularweight(or number of bp) of the donor fragment, and on the marker itself.

There are low efficiency and high efficiency markers. Low efficiency markers

are recovered at low frequencies and high efficiency markers at high

frequencies. The efficiency is a result of different mismatch repair

probabilities (in strains where transformation takes up only single stranded

DNA).

Furthermore reciprocal transformation do not yield the same transformation

frequencies, eg if there are two alleles Z and z, transformation and recovery

is not the same if Z is the donor and z the recipient as with z the donor and Z

the recipient allele because of differences in mismatch repair. Therefore, in

transformation recombination frequencies between donor and recipientdont depend on, or correlate with distancebetween markers.

But the probability of two markers to be transformed (and detected)

togetheris increased if they are located closely together on the same DNA

fragment. Hence cotransformation frequencies go up the closer two markersare.


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When DNA from donorbacteria is isolated, most of it is also broken into

hundreds of random size smaller fragments. If such DNA is used to

transform highly competent recipient cells, the probability or frequency of

transformation of most genes is about one cell per 1000.

If two genes x and y are separated and the distance between them is

larger than the length of most of the transformed fragments, that is, if the two

genes are rarely observed on the same DNA fragment , then the

transformation frequency of both together is the square of the individual

frequences (10-3)2or 10-6. However, if the two genes are located so closely

to each other such that they often end up on the same donor DNA

fragment, then the frequency of cotransformation is much closer to 10-3,the frequency of transformation of a single gene alone. Thus co-

transformation of two genes at a high frequency implies that they are

located closely together.

Example: if genes y and z and genes z and x can be cotransformed but not y

and x, then the order of genes must by y z x.

In general, if the size of the DNA fragments is controlled within a reasonably

narrow range, then one can relate the cotransformation frequency with the

distance between the genes. I.e. if one measures the cotransformation

frequency as a function of the molecular weight (or length of the DNA) one candetermine the distance between the genes roughly.


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If a and b, and b and

c can be

cotransformed but not

and c, that means the

order of the genes

must be a,b,c.

However,

cotransformation

frequencies (CF) are

not the same as

recombinationfrequencies. The CF

is determined mostly

by the size of the

fragment and the

likelyhood of strandbreakage of bacterial

DNA, rather then than

chiasma formation of

homologous

chromosomes.


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Distance is not the only variable in cotransformation frequency:

At high DNA concentrationsrelative to the number of competent cells, more

than one DNA fragments binds each cell and the cell may take up several

fragments, this may appear as if two genes are cotransformed and lead to the

wrong interpretation of the data. Therefore, one may have to perform a dilution

test. If following dilution, the second gene B is cotransformed with the other

gene A, at a similar frequency as gene A or B alone then the two genes are

closely linked. Alternatively, if the cotransformation frequency drops severely

following dilution, then the two genes are not (closely) linked.

If the genes are not closelylinked then a tenfold decrease

in DNA concentration should

lead to a hundred fold

decrease in cotransformation of

both genes, while the drop off

should be by a factor of 10 forthe individual genes. If the two

genes are closely linked, then a

10-fold dilution should lead to

only a 10 fold drop in

cotransformation.


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Problem: Because transformation is performed with DNA fragments and

because the genetic markers used for gene mapping may be far apart, there

will be problems in the determination of neighboring genes in such areas

(where there are only few or no markers) because one may not be able to

observe cotransformation. As a result, mapping a circular bacterial genome

through cotransformation produces areas where the linkage between genes

is determined (linkage groups) and gaps between those linkage groups

where there are not enough closely spaced markers.

(It is possible to close those gaps by taking cultures in which DNA replication

is synchronized and determining the sequence of gene replication from an

origin of replication, this involves separating newly synthsized DNA from oldDNA by 15N labeling. )

Linkage group 1 linkage group2

No cotransformation between genes from these two linkage groups

because the distance between genes/markers is too large.

I it l id t f


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In vitro plasmid transfer:

How does one transfer a non-transmissible plasmid to a specific host cell?

One purifies the plasmid DNA and transformsthe strain of interest with it,

applying a genetic selection, usually antibiotic resistance. Transformation

means spontaneous uptake of the DNA by the recipient bacterial strain. Some

bacterial strains are naturally transformable, however, the strains most

commonly used in molecular biology need to be prepared to be come

transformation competent.

A common method of making E. coli chemically competentis

through hypotonic shock in the present of divalent ions, such as Ca2+, Mn2+

and Mg2+. Early log phase cultures are centrifuged, and resuspended inhypotonic solution. When DNA is added it forms a complex with the Calcium

that adsorbs to the cell surface. Cells are then warmed/heat shocked, this

transport into the cell.

Alternatively cells can be made permeable with electroporation. The cells are

exposed to an electric field and and electric discharge that polarizes themembrane. The voltage potential across the membrane forms transiently

small pores, making the cell permeable, allowing macromolecules such as DNA

to enter.

In vitro transformation uses double stranded DNA, and because it uses self-

replicating plasmids it does not require recombination. However the size of

DNA that can be taken up by bacteria in that way is limited.

C j ti


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Conjugation

Conjugation is the direct unidirectional transfer of DNA from on bacterial

cell to another, in most cases plasmid DNA, although in some species

chromosomal transfer can also occur.

Conjugation can easily be demonstrated among Enterobacteria and other

Gram-negative bacteria. But gram positives like Streptomyces also

possess conjugation systems.

Conjugation is not necessarily confined to members of the same species,

therefore, it is another avenue for horizontal gene transfer across

taxonomic boundaries. As a result plasmids that are present in the normal

gut flora can be transmitted to infecting pathogens, which then can becomeresistant to a range of antibiotics.

Mechanism: As already mentioned, it requires a donor straincontaining

a plasmid that carries the genes required for promoting DNA transfer. In

E.coli and other Gram-negative species, the donor cells carry pili, whichvary in structure length, flexibility etc., depending on the plasmid. The pili

bring the cells into contact and then a channel or pore is made through

which the DNA is transferredfrom donor to recipient.

Apparently this mechanism has many things in common with protein

secretion systems used to deliver bacteria toxins directly into host cells.


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Some large plasmids contain

genes that enable plasmids to

be transferred between cells. In

E. coli

For example there is a largeplasmid called F factor (F like

ertility). Cells containing F

actor are F+, those lacking it are

F-. Transfer of the F plasmid is

mediated through a tube likestructure, called the pilus, during

conjugation. The F plasmids or

conjugative plasmids contain

about 20 genes that are required

or pilus assembly and generansfer. Most smaller plasmids

dont contain those genes,

however, by means of

recombination with the F

plasmids, they can tag along

ith the conjugative plasmids.


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Conjugation is the process in which DNA is transferred from a donor cell to a recipient cell, by cell to cell contact

and DNA transfer through a conjugation tube (or F- pilus). It occurs in many species, originally discovered by

J. Lederberg. The process is controlled by and is dependent on a set of genes that is encoded in an

independent circular DNA molecule (F plasmid, autonomous state) in F+cells, or it can be integrated in the

bacterial chromosome in hfr cells (high frequency recombination). When an F+donor cell conjugates with and

F- recipient cell, only the F factor is transferred, both the donor and recipient cells end up F+, since the plasmid

is replicated by a rolling circle mechanism during transfer. If a few F+cells are mixed into a majority of F-cells,most of them end up being F+. (F stands for fertility.)


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Conjugation is a replicative process that transfers a copy into the recipient


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Conjugation is a replicativeprocess that transfers a copy into the recipient,

but also leaves another copy in the donor cell. Thus both cells are donors

after the mating, hence this process can lead to an epidemic spread of the

plasmid throughout a bacterial population.

Plasmids that can mediate the complete DNA transfer process are called

conjugative plasmids. In some cases the conjugative plasmids can also

promote (mobilize) the transfer of a second otherwise non-conjugative

plasmids from the donor cell (donation).

ColE1 is an example of a plasmid that has the genes needed for DNA transfer,

but not the genes required for mating-pair formation. Mobilization involves the

mob gene which encodes site specific nuclease that nicks DNA at the bomsite (=oriT, the origin of transfer).

Not all mobilizable plasmids have the mob gene. In that case, the Mob

nuclease has to be provided by the conjugative plasmid for mobilization.

However, no mobilization happens without a bom site on the second

mobilizable plasmid. Removal of the bom site from plasmid makes it non-mobilizable, and non-transferrable through conjugation.

The primary goal of conjugation is transfer of the conjugative plasmid to the

recipient, converting the recipient into a male donor cell, spreading the

plasmid through the whole population of bacteria. However in some cases

some types of plasmids can also promote transfer of chromosomal DNA.


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bom=oriT

In the case of the F plasmid the transfer of chromosomal sequences


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In the case of the F plasmid, the transfer of chromosomal sequences

involves prior integration of the (conjugative) F plasmid into the host

chromosome. As a result a part of the host chromosome is transferred, as

part of the attempt of the F plasmid to transfer itself. (In other cases

chromosomal transfer occurs without stable association between the plasmid

and the host chromosome).It is however impossible for the complete copy of the chromosome to be

transferred because DNA transfer during conjugation is a slow process that

would take about 100 min for the whole chromosome. Matings rarely last

that long, and therefore, the process tends to get disrupted before the

transfer of the genome is complete. The mating lasts long enough for aplasmid of 40-100kb which requires 1 min. to be transferred, but not for the 4

mb of a chromosome which would require 100 min.

Hfr strains (high frequency of recombination).

Hfr strains arise by integration of the F plasmid into the bacterialchromosome. As in the conjugative plasmid the starts at an origin of transfer

of the plasmid and is determined by the site of insertion of the plasmid within

the genome. The direction of transfer is determined by the orientation of the

plasmid inserted. Hence different Hfr strains of the same species have

different origins of transfer and different directions, depending on the

particulars of the site and direction of insertion into the host chromosome.

The F factor can integrate into the bacterial chromosome by site specific recombination to create


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The F factor can integrate into the bacterial chromosome by site specific recombination to create

Hfr cells (High frequency recombination.) Recombination occurs by site specific homologous

recombinationbetween insertion sequences (IS) present on the plasmid and the chromosome.

IS sequences originate from transposons.

After conjugation between Hfr cells and F- cells the transferred DNA will not become circular and is


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B-chromosome

After conjugation between Hfr cells and F cells the transferred DNA will not become circular, and is

not capable of further replication, because most of the time the transferred DNA does not contain

all the genes necessary for conjugation and autonomous replication, as transfer usually gets

interrupted prior to completion of transfer.

Bact.

Chromosome

in Hfr cell

The DNA transfer is

controlled by the integrated

F factor, and is initiated on

the Hfr chromosome at the

same site as in the F

plasmid. A part of F DNA is

transferred first, followed by

chromosomal genes and the

remainder of F last,

however, the latter rarelymakes it into the recipient

cell, hence the recipient

remains F-.


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Because it involves transfer of a bacterial chromosome (4600kb) conjugation

between Hfr cells and F- cells would take 100 min, vs. 2 min with F+ cells (100kb).

Usually only a fraction of several 100 genes of the chromosome is transferred before

conjugation is disrupted and the cells separate.

The recipient usually remains F- because separation takes place before the entire

chromosome is transferred. Some regions of the transferred DNA becomes

integrated into the donor cells by homologous recombination. Where

integration occurs, cells become recombinant, however the donor genomes remain

unchanged. Eg. Donors are Hfr Leu+, recipients F- leu-, recombinant recipients will

be F-Leu+. Because recombinants occurs in only a small fraction of all the bacteria,

selectable markers need to be employed to identify recombinants, and

counterselection is used to get rid of donor cells. Eg. The the transferred selectable

marker for recombinants could be Leu+, while the counterselection marker employed

could be Str-s (streptomycin resistance) which would be only present in recipients.

Hfr matings can be used for bacterial chromosome mapping. In this case, the

genetic map is based on transfer order, not on meiotic recombination. The geneticmap is obtained by interrupting the DNA transfer during the mating (i.e. after Hfr and

F-cells have been mixed) with a blender like device. The earliest time at which a

particular gene is transferred (time of entry) , and at which mating disruption no

longer interferes with the appearance of recombinants, that time denotes the relative

position of that gene on the chromosome, since transfer is unidirectional. Many

selectable marker genes have been mapped in that way.


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Interrupted mating technique

The number of recombinants increases with length of time of mating.


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g g

Each marker has time of entry before which no recombinants are detected.

Each curve has a linear region that can be extrapolated back to the time axis, defining the time of

entry. The period of increasing recombination is due to a variation in the initiation of conjugation.

The number of recombinants of each type reaches a maximum or plateau, the value of which

decreases with successive times of entry.


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The genes that can be mapped with the interrupted mating technique, depend

on the location and the direction in which the F-plasmid integrated into the

host genome.

Different F- strains contain different alleles, thus adding more information to the map

(C). Different Hfr strains differ in their location of F and hence in origins and direction

of transfer, since F can integrate at multiple sites. Combining the overlapping maps

obtained with different Hfr strains yields a composite map, that is circular because the

E. coli chromosome is circular.


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Circular map of E.coli. Map

distances

are given in minutes. The total map

length is 100 min. For some loci

that encode related gene products,

the map order of the clusteredgenes is shown along with the

direction of the transcription and

length of transcript, (black arrows).

The purple arrow heads show the

origin and of transfer of a number of

HFR strains. For example, Hfr

transfers the thr early, followed by

leu and other genes in a clock-

wise direction.

Complete F plasmids are occasionally


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p p y

excised. Aberrant excision creates an

Fplasmidthat contains a fragment of

chromosomal DNA. By the use of

different Hfr strains with different

origins of transfer, Fplasmids carrying

segments from many regions of thechromosome have been isolated. F

plasmids dont require packaging, and

their size is less restricted than that of

phage. Any recipient cell is partially

diploid (meroploid)for the

chromosome segment carried on the

plasmid.This is useful for testing dominance,

and gene dosage, as well as

complementation. F particles may

carry selectable markers such as thr+

or leu+ that can recombine with the

recipient genomewhile the rest of the Fparticle may be lost. Recombination

mediated by F particles is called

sexduction or F-duction.


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Mapping closely linked genes in bacteria:Interrupted mating experiments provide

approximate map distances for genes that are several map units apart. The resolution is

, however too coarse for genes that are one or two minutes apart. In order to resolve

the relationships between closely linked genes, three-point crosses are necessary.

With one major difference the logic of three-point crosses is mostly the same as the logicobserved in diploids.

With rare exceptions, recombination in bacteria takes place between fragments of a

donor chromosome carried on a transferred (exogenote) piece of DNA (by

transduction, transformation or sexduction,) and a recipient chromosome, theendogenote. Recombination requires a double crossover since bacterial

chromosomes are circular. Therefore, with merozygotes or partially diploid cells, a

three factor cross can be performed in two ways:

(i) double mutant donor X single mutant recipient, (ii) single mutant donor X double

mutant recipient. The result of such a reciprocal cross can be used to order the

markers involved.

Consider: a+b1+ b2 donor X a b1b2

+recipient


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1 2 1 2 p

a b1b2+ donor X a+b1

+b2recipient

If the order is a b1b2 then a+b1

+b2+recombinants will occur with similar frequency

because only one double cross over is required for both crosses to get a+b1+b2

+

recombinant, assuming that all markers are conditionally lethal:

cross1 cross2

Donor/ exogenote

recipient/ endogenote

a+ b2 b1

+

a b1 b2

+

a b1 b2

+

a+ b1+ b2

a+ b1+ b2

a b2+ b1

a+ b2

b1+

a b2+ b1

If the order is a b2b1then the requirements are different:

Donor/ exogenote

recipient/ endogenote

Two double crossovers

low frequency. One exchange/double crossover, higher frequency.


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If the order is a-b-c then the frequency of a+b+c+ in cross 1 will be approx. equal to the

frequency of a+b+c+in cross2. If the order is a-c-b, then the frequencey of a+b+c+ in

cross 1 will be much lower than the frequency of a+b+c+in cross 2.

donor recipient

Transduction


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Generalized Transduction:

Transfer of bacterial DNA from

one bacterial cell to another by

phage particles is called

transduction. Generaltransduction is the result of

accidental packaging of

(nuclease fragmented) bacterial

DNA (from any part of the

bacterial chromosome) into

phage particles.

In specialized transductionthe

phage particles contain both

phage DNA and bacterial DNA in

one molecule, however, the

bacterial DNA is derived from aparticular chromosomal region

close to the phage integration

site.

Nuclease catalyzed

fragmentation of b.

chromosomes

1in 106

Transducing particle

contains about 50 genes

After recombination leu+, will survive on selective growth medium

Demonstration of


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Demonstration of

linkage by cotrans-

duction.

The transduced

Fragment contains

about 50 genes.

Whether 2 genes

are cotransduced or

not depends on the

distance.

Cotransduction of

bio and leu markers

can be detected by

growth on selectivemedium.

Gal+and bio+are

cotransduced about

12% of the time.

Leu and gal

cotransduction does

usually not occur

because the

distance is too far.

Cotransduction

frequencies make it

possible to construct

linkage maps.

Three factor crosses

can be applied asdescribed above.

Temperate phage cycle.


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Loss of prophage

(curing)

Nonlysogenic

cell

lysogenic

cell

lysogenic

response

lytic

response

induction

Vegetative

phase

Transduction requires a lysogenic, rather than a lytic phage.

Lif l f (l ti ) b t i hMapping in phage


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Life cycle of a (lytic) bacteriophageMapping in phage.


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Phagemaps can be constructed

f h bi ti


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from phage recombination

experiments.

Early mutation mapping

experiments suggested

T4 phage mapped to 3

different clusters. Allclusters showed linkage.

Three point crosses indicated

that the map could be circular.

In T4 genes are also clustered

according to function.


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The wildtype phage T4 can be grown in either E.coli strain B or K12(l). T4 with

mutations in the rII gene can be grown as large round plagues in B but not in K12(l)

which is a lambda lysogen. If the B strain is infected with two different phageT4 rII

mutant recombination occurs (somewhat rare) from which rII+ phage results that can

grow on K12(l). Mutagenesis yields a large number of rII mutations. Mutations that failto recombine with several known point mutants (that were known to recombine with

other point mutants) these are taken to be deletion mutants.

The deletion mutants eliminates a part of the phage genome. The deletions can be

used to order maps obtained by point mutations. If a cross between a point mutant and

a deletion mutant produces a wildtype recombinant, that means the point mutant wasoutside the region that was missing in the deletion (eg. Mutants 1,2,3.

If a deletion and a point mutant overlaps with the deletion, then wildtype recombinant

progeny cannot be produced by crossing the two..

No recombinants in this interval between the deletion mutantand point mutants 5 and 6

Deletion mutant

1 2 3 4 5 6 7


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These studies provided important experimental support for the following concepts:

Genetic exchange can take place within the coding sequence for a gene, at almost

any nucleotide.

Mutations are not distributed evenly across a gene. Mutational hotspots exist, i.e.sites that are much more likely to be mutated.

Also this mutation analysis helped distinguish between three different definitions of

gene as a unit of function or cistron: a stretch of DNA encoding a functional protein.

Complementation test : When mutants in rIIA and rIIB were combined to infect E. coli

simultaneously, normal numbers of phage were produced without recombination.I.e. rIIA and B function independently of each other and can therefore complement

each other. Different mutants within the same gene eg. rIIA cannot complement. ( A

functional protein may be encoded by two subunits locaten on two different genes.)

Other meanings of gene: (1) unit of genetic transmission that participates in

recombination. (2) unit of genetic change or mutation. Both can correspond toindividual nucleotides in a gene.

A virulent phage undergoes only the lytic cycle, in which the phage takes over the cell

to produce only phage protein and replicate phage DNA In the lysogenic cycle the phage


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to produce only phage protein and replicate phage DNA. In thelysogeniccycle the phage

genome is integrated into the bacterial genome, where it replicates along with the bacterial

genome as prophage. The bacterial cell is now called lysogen, eg. K12(l). The phage DNA

can be activated and excised under certain conditions that damage DNA, such as UV radiation.

Lysis follows.

At this point the genome of lphage has been analysed extensively, both in therms ofsequence and functionally l genes exhibit extensive clustering by function eg head


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sequence and functionally. l genes exhibit extensive clustering by function, eg head

proteins, tail proteins, DNA replication, recombination , lysis immunity, etc.

Clustering also occurs in terms of the timing of product synthesis, eg early and late

genes.

Interrupted mating of E.coli cells nonlysogenic and lysogenic for lphage, revealed that

the lgenome is inserted between the gal and bio genes of E. coli chromosomes.

I.e. the presence of prophage increases the physical distance between gal and bio,

since gal and bio can be cotransduced by P1 phage on a nonlysogen, but not on a

lysogen for l.

Genetic mapping of the prophage yields a permutation of the genetic phage map derivedfrom standard phage crosses.

The DNA of phage l is a linear


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p g

molecule with complementary

cohesive ends (cos) each 12b, such

that they can recombine and form a

closed circle upon ligation.

Circularization is required for boththe lytic and lysogenic cycle.

The site of breakage and rejoining inbacterial and phage DNA are called


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The geometry of integration

and excision of phagel. The

phage attachment site is POP.

The bacterial attachment site is

BOB. The prophage si flanked

by two hybrid attachment sitesdenoted BOPand POB.

attachment sites. They have three

segments each. The central

segments are identical in their

nucleotide sequence for both sites.

POP is located near the middle of the

linear form of the phage. A phageprotein, integrase, recognizes the

attachment sites and catalyzes site

specific recombination between them.

Hence the phage map is a

permutation of the prophage map.

Upon integration the often only one

phage protein is expressed in the

lysogen, the phage repressor, whichprevents other phages of the same

kind from infecting the lysogen,

conferring immunity from lytic

infection.

The prophage can becomeactivated to excise and undergo


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activated to excise and undergo

a lytic cycle upon inductionby

DNA damaging UV light or

environmental agents. Excision

requires two enzymes,

excisionase and integrase, thelatter for site recognition.

In about 1out of 106or 107

excision events errors occur, in

which the sites of breakage and

rejoining are displaced.

Sometimes such an aberrant

molecule can replicate and getspackaged. The aberration

occurs either to the right or the

left of the molecule, including

either bio or gal genes. The

resulting phage particles are

called ldgal or ldbio. They are

called specialized transducing

phages because they can

transduce only a limited number

of genes, such as

the gal or bio genes.

Because specialized transduction particle are deficient in some genes, they require wildtype lhelper phage to integrate and replicate. Wildtype l phage is required for integration because its


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p p g g p yp p g q g

integration provides two hybrid attachment sites homologous to the one present in ld phage. Also

wildtype lphage provides genes that are missing within the ld phage. The segment of donor DNA

and the phage chromosome are added to the recipients chromosome, producing a partially

dipoidtransductant.

Usually when lysogenic phage is excised, few defective transducing particles are formed byaberrant excision, hence the resulting lysates are called low frequency transducing lysates.

If enough wildtype phage is present the recipient cells are also infected with wildtype phage that

integrates at the normal attachment site, producing double lysogens, (carrying l+andldgal). The

resulting transductants are thus gal-/gal+ partial diploids[and are also called gal+/gal-

heterogenotes, containing gal+exogenote(the donor DNA fragment) and gal-endogenote

(recipient chromosome)]. The partial diploids are unstable transductants because can exit thechromosome and be lost.

In addition,ldgal-l+double lysogens can be induced to lyse with UV light , producing 50% ldgal

and 50% l+HFT (high frequency transduction ) lysates. HFT lysates facilitate genetic analysis by

via specialized transduction by dramatically increasing the frequency of transduction events. Gal

can be used as a selection marker, since only gal+ cells can utilize galactose as a carbohydrate

substrate. In general specialized transduction can be used to map phage attachment sites and the

genes that are close to the attachment site. In general specialized transduction is not the most

useful technique encountered in prokaryotic genetics.


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phage bacteria

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