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Chapter
6. Genetic Transfer and Mapping in Bacteria and Bacteriophages.
Bacteria
reproduce asexually so researchers rely on genetic transfer instead
of crosses.
A
segment of bacterial chromosome is passed from one bacterium to another.
Used
to map gene locations along the single circular chromosome.
We
will also discuss genetic mapping in bacterial viruses or bacteriophages.
There are
three naturally occurring ways that genetic material can go from one
bacterium to another: conjugation, transduction and transformation. Conjugation:
Transduction: A virus
that infects bacteria (bacteriophage) transfers genetic material from one
bacterium to another. Transformation: Genetic material from one cell is released into the environment (death) and another bacterium takes it up.
Joshua Lederberg and
Edward Tatum were the first to demonstrate the natural ability of bacteria
to transfer genetic material.
They were studying a
strain of E. coli called B-M-P+T+ that had certain growth
requirements.
Required
the vitamin biotin (B), the amino acid methionine (M); and
did not require phenylalanine (P) and threonine (T).
Another
strain B+M+P-T- had the opposite requirements.
These
differences in requirements were the result of differences in DNA.
The B-M-P+T+ has
defects in a gene encoding for an enzyme involved in the biotin and
methionine biosynthetic pathways.
The B+M+P-T- has
defects in a gene encoding for an enzyme involved in the phenylalanine and
threonine biosynthetic pathways.
If either of these
strains are grown and plated at a concentration of 109 cells on
plates that do not have amino acids or biotin they will not grow. However, if you mix the two cells together and plate 109 cells approximately 100 cells will grow.
Lederberg and Tatum
surmised that genetic material was being transferred between the two cell
types.
This allowed the two
bacteria to form new bacteria that were B+M+P+T+. This was further established with the U-tube that would only allow small molecules to pass between the two halves of the tube.
Only about 5% of
bacteria can act as donors.
Donor strains
contain a small circular segment of DNA (plasmid) known as F
(fertility) factor these strains are denoted F+.
The sex pili act as
an attachment site and allows for physical contact between F+ and F-
strains.
One strand of the F
factor plasmid is transferred to the recipient cell.
This
transfer begins at a site called the origin of transfer.
Hfr
strains
of E. coli contain F+ factor integrated into the chromosome and during
conjugation the bacterial chromosome is transferred from the donor to the
recipient in a linear fashion.
These strains will
also transfer chromosomal genes to the recipient cell.
Hfr stands for high
frequency of recombination.
F factor has become
integrated into the chromosome. The origin of transfer is the first place that is cut and readied for transfer.
From this starting
point all the bacterial chromosome enters the F- recipient cell in a
linear manner.
Once inside the F-
bacterium this DNA can swap or recombine with the recipients own
chromosome.
This recombination
may result in a new combination of alleles. Ex: A recipient strain that is lac- (unable to metabolize lactose) and pro- (unable to metabolize proline) The donor cell in this case is lac+ and pro+.
Depending on the
length of mating the recipient cell could become lac+ or both lac+ and
pro+.
Since the
chromosomal DNA is passed to the F- cell in a linear fashion a longer the
mating time will result in more chromosomal DNA being passed to the
recipient cell.
In this example lac
is always transferred before pro due to the orientation of the origin of
transfer. Different Hfr strains have different locations for their origins of transfer and different orientations.
Wolling
and Jacob used Hfr matings to map genes along the E. coli
chromosome.
Used a blender at
speeds that separate conjugating bacteria but do not destroy the cells.
The
rationale behind their study was that the time it takes for a gene to be
transferred to the recipient cell is directly related to their order on
the chromosome. They realized since the chromosome is transferred linearly interrupting conjugation at different times would lead to various lengths of the Hfr chromosome entering the recipient cell.
By determining which
genes were transferred during quick matings and which were transferred
during slow matings they could order the genes along the E. coli
chromosome.
The donor strains
genetic composition.
T+:
synthesizes threonine.
L+:
Synthesizes leucine.
Azs:
Sensitive to the toxic chemical azide.
T1s:
Sensitive to bacteriophage T1 infection.
Lac+:
Metabolizes lactose and use it for growth.
Gal+:
Metabolizes galactose and use it for growth.
Strs:
The recipient strain
has the opposite genotypes for these genes: T-, L-, Azr, T1r,
lac-, gal-, and strr where r = resistant.
They knew that the T
gene was transferred first and the L gene second both after 5-10 minutes
of mating.
The goal of the
experiment was to determine the times at which Azs, T1s,
lac+ and gal+ where transferred.
Strs
was not examined because streptomycin was used to kill the donor strain.
Hypothesis:
The chromosome of the donor strain is transferred in a linear fashion to
the recipient strain. The order of the genes along the chromosome can be
deduced by determining the time various genes take to enter the recipient
strain.
How did
they test for gene transfer?
First took mated
cells and plated them on plates lacking threonine and leucine but had
streptomycin.
This
made sure that neither the original donor or recipient strains could grow.
How?
The recipient cells
that had received the T+ and L+ genes would be able to survive. In order to determine the order of transfer of the Azs, T1s, lac+ and gal+ genes the experimenters picked colonies from the above plate and streaked them on plates that had azide or bacteriophage T1, or on minimal plates that contained either lactose or galactose as the sole energy source.
These plates are
then incubated overnight and analyzed for growth.
Recipient
cells that have received the Azs gene cannot grow on azide.
If
the recipient has received the lac+ gene it will grow on plates that only
have lactose as an energy source.
If
the recipient has received the gal+ gene it will grow on plates that only
have galactose as an energy source.
Interpretation
of data:
After the first
plating all survivors would be F- cells that have received T+ and L+
alleles and F- cells are already resistant to streptomycin.
Transfer
of T+ and L+ occurred after 10 minutes.
Each of the
surviving T+,L+ colonies were then tested for the transfer of the
remaining genes (Azs, T1s, lac+ and gal+).
Where
the colonies sensitive to killing by azide, infection by T1, able to
utilize lactose or able to utilize galactose?
After
how much mating time did each of these phenotypes appear?
A pattern emerged.
The
gene conferring sensitivity was transferred before the gene conferring
sensitivity to T1 phage infection.
The
transfer of these two genes was followed by lac+ and then finally gal+.
From this data a map
can be generated. Studied many Hfr strains and their data was consistent with a circular E. coli chromosome.
Conjugation
studies have resulted in a detailed genetic map of the E. coli
chromosome.
Mapped 1,000 genes
of the E. coli chromosome.
The arbitrarily
assigned starting point is at 0 minutes and the chromosome is 100 minutes
long.
Which is the time it
takes to transfer the complete Hfr chromosome during mating. Maps generated from bacterial conjugation studies are are illustrated using the unit minutes.
Bacteriophages
can also transfer genetic material from one bacterium to another via
transduction.
Bacteriophages
contain genetic material surrounded by a complex protein coat.
Bacteriophages
attach to the surface of a bacteria and inject there genetic material. Lysogenic cycle: Phage DNA inside the bacterium integrates into the chromosome (creates a prophage) of the cell and is reproduced along with bacterial reproduction.
Lytic cycle: The
bacteriophage directs synthesis of multiple copies of the phage genetic
material and coat proteins.
These
components are assembled as phages which then lyse the host cell and
infect other cells.
Cells can begin in
the lysogenic cycle and enter the lytic cycle at a later time.
The
prophage exits the bacterial chromosome initiating the lytic cycle.
When certain phages
enter the lytic cycle the bacterial chromosome becomes fragmented.
These fragments are
sometimes packaged inside bacteriophage coat proteins and delivered into a
new bacterium.
These fragments of
DNA are then free to integrate into the new cell resulting in a new
combination of alleles. This type of transduction is called generalized transduction.
If two
genes are close together on the bacterial chromosome they may be packaged
in a single bacteriophage and transferred as a single unit, cotransduction.
The likelihood of
cotransduction depends on the distance between the two genes.
A bacteriophage
cannot physically carry DNA fragments larger than 1-2.5% of the bacterial
genome.
Cotransduction
frequency = (1-d/L)3 d= distance between the genes in minutes and L= size of chromosomal pieces.
Bacteria
can transfer genetic material by transformation.
The DNA fragment may
then incorporate into the recipient cell making a recombinant bacterium.
Competent cells are
special bacterial cells that can undergo transformation.
These
cells are influenced by ionic concentration, temperature, and nutrients in
their environment.
A
fragment of DNA binds to the surface of the bacterial cell.
The DNA is then
cleaved by an endonuclease to a manageable size.
The DNA interacts
with proteins from the cell surface and either one or both strands is
brought into the cell.
This DNA then may be
incorporated into the recipients chromosome.
If a single strand
has been taken up and is recombining into the chromosome sometimes a heteroduplex
may form.
Heteroduplex:
region of mismatch.
These are fixed by
DNA repair enzymes and can be used to repair mutations in bacteria.
Intragenic
Mapping of Bacteriophages.
Viruses
are not living, however they do have traits.
They are a product
of their genetic material.
We will
focus on bacteriophage T4 which has several dozen genes that make
up its genetic material.
These genes encode
for proteins necessary for the viral life cycle.
Proteins
involved in coat assembly, lysis etc..
Example:
Tail fiber proteins that allow it to attach to bacterium. »
Bacteriophage
specific proteins.
Seymour
Benzer (early 1950s) did much early work on bacteriophage DNA that led
to a basic understanding of all genetic material.
Intragenic
mapping (fine structural mapping): Is
the mapping of the sequence differences between two alleles of the same
gene.
Intergenic
mapping figures the distance between two genes.
Lytic
bacteriophages produce viral plaques.
Lytic phages cause
lysis of the host cell so the newly formed phages can infect more cells.
This
cycle causes areas on a plate where all the bacterial cells have been
lysed.
These
areas appear as plaques (an observable clear area) on a lawn of bacteria.
Mutational analysis
of phages resulted in phages that would cause different plaque
morphologies.
rII T4 phage
resulted in a few plaques that were abnormally large. This phage variant forms different plaques depending on the bacterial strain it infects.
Complementation
can reveal if mutations are in the same gene.
How can two
different mutations affect one trait?
One way is that the
two mutations may be in the same gene.
What
is the other way? Example?
Phage mutations can
be examined by coinfecting cells with two phages that have mutants that
affect the same trait.
What
would happen if the mutants were in the different genes?
The
same gene?
Complementation:
The mutations were in different genes allowing one wild-type gene from
each phage to complement the mutant gene of the other phage. »
Able
to form plaques.
Noncomplementation: Coinfection does not allow for plaque formation since both
phages had mutations in the same gene. »
Cistron
= gene. »
His
studies on rII phage resulted in the identification of two genes involved
rIIA and rIIB.
Intragenic
maps were generated using data from recombination within the rII region.
At an extremely low
rate two noncomplementing strains can produce a plaque.
Cannot
be homoallelic mutants (the same mutation).
How?
Crossover
between the two mutations within the gene.
Probably
as infrequently as 1 in 100,000.
Needs
to be a rapidly reproducing organism. Benzer used this rarely occurring recombination event to measure the distance between the two mutations (intragenic distance).
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