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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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