University of Glamorgan
Module: BI 208 Genetics
Lecturer: Dr Jill Williams
Essay on Bacterial Interrupted Mating Experiment
The Interrupted
Mating Experiment technique with bacterial cell was worked out by two French
geneticists, François Jacob and
Elie Wollman in the late 1950’s. They were trying to demonstrate the mechanisms
of gene transfer using Escherichia Coli.
This technique enable scientist to
map, for the first time, any genome longer than that of a phage or a virus.
In their experiment, Jacob and
Wollman mated an Hfr donor cell
(which contains the F factor
integrated into the main bacterial chromosome) with an F- recipient cell, following the procedures of the
conjugation experiment performed earlier by Hershey and Chase.
The genotypes of the two E. coli strains involved was:
Donor – HfrH: thr+ leu+ azir tonr lac+ gal+ strs
Recipient – F-: thr- leu- azis tons lac- gal- strr
(The superscripts “s” mean sensitive to, “r” resistant to, “+” able to synthesise or metabolise the compound, and “-“ unable to synthesise or metabolise
that compound).
The Hfr strain used was the E.
coli HfrH (where H strands for
Hayes, another scientist who had an important role in the discovery of the
bacterial mating mechanism)
This strain was prototrophic (wild
type strains that are able to synthesise all the essential nutriments) and
sensitive to the streptomycin antibiotic. The F- strain carries the gene for streptomycin resistance
and a number of mutant genes, which cause it to be auxotrophic for threonine (thr-) and leucine (leu-), sensitive to sodium
azid (azis) and to
infection by bacteriophages T1 (tons),
and unable to ferment lactose (lac-)
and galactose (gal-).
The two strains we mixed in
nutrient medium and incubated at 37(C to allow conjugation to start.
In the beginning of conjugation,
the integrated F factor is nicked at
the origin and replication takes place by the rolling circle mechanism. The
first genes to be transferred are those of the F factor. The bacterial genes close to the site of plasmid
insertion, can also be sequentially transferred to the recipient cell if the
conjugation process lasts long enough.
The donor and recipient cells alls
are physically linked through a sex pili, which is synthesized by the donor
cell. The pili is a very fragile structure and break easily. While the bacteria
conjugate they jiggle around in a natural Brownian motion, which put the pili
under physical stress and breaks it. This is why in nature only an average of
25-30% of a bacterial chromosome is transferred to the recipient cell.
The experimental design of Jacob
and Wollman involved the use of a kitchen blender* to break the matting cell
apart at various times after the beginning of conjugation. This stopped the
transfer of DNA. The longer the genetic transfer was allowed to take place, the
more genes were transferred. The genes that are passed to the recipient cell
become incorporated into the main bacterial chromosome by two crossover events.
The resulting recombinants are partially diploid. This means, that they are
diploid for the genes that were transferred from the donor cell and haploid for
all other genes.
Figure 1
–Interrupted Mating Experiment performed by Jacob and Wollman (a) procedure,
(b) results
Once the transfer was stopped the
cells were removed from the mating mixture and then were plated on a selective
medium, specially conceived to allow only the growth and division of the
recombinant cells. The HfrH and F- cell should not be able to
grow. In this particular case the medium contained streptomycin that killed the
HfrH cells and lacked threonine so
the F- cells could not
grow. Other appropriate media were used to test the appearance of certain donor
genes among the selected thr+
leu+ strr transconjugants.
In this experiment the selected
marques were thr+ leu+
strr and the azir
tonr lac+ and gal+
genes were the unselective markers. The time of transfer of the first selected
genes thr+ and leu+ was defined as time zero
(measured in minutes).
The data collected from this
experiment is shown in figure1.
From these results it is possible
to determine the order of transfer of the unselected gene markers as a function
of time. The first gene to be transferred was the one for azide resistance (azir), which is the result of
a mutation in the gene sec A that is
normally involved in protein secretion. This gene appeared at about 8 minutes.
The second gene to be transferred,
the tonr appeared at 10
minutes. The resistance to bacteriophages T1
is determined by a mutation in the fhuA
gene which codes for the outer membrane receptor for ferrichrome, colicin M and phages T1, T5 and phi80.
At about 17 minutes the lac+ gene was transferred
followed by the gal+ at
approximately 25 minutes. These two genes code for the lactose and galactose
metabolisms respectively.
From the analysis of the
appearance rates of each gene, which are indicated by the slope of the curves,
and the maximum frequencies obtained for each recombinant type (the height of
the plateau) it is possible to conclude that:
·
As the conjugation time
increases, the rate of appearance and the maximum frequencies of recombinant
decrease.
·
The rate of transfer from
one mating couple to another is not constant because cells are not synchronised,
that is they do not initiate DNA transfer all at the same time.
·
The later the gene enters
the recipient cell, the smaller is the maximum frequency of recombinants
because the probability of the mating cells breaking apart as the result of the
Brownian motions increases with time.
The time intervals between the
appearance of each gene is used to determine the distance between them (the
distances being measured in minutes).
From this information we can
conclude that gene transfer occurs in a linear way, and that the genes that are
far from the origin tend not to be transferred to recipient cell because of the
higher probability that be mating pair will break apart before their transfer
can take place. So being the F-
cell only very rarely receives the entire F
factor (part of which is at the other end of the bacterial chromosome), thus
becoming an Hfr cell.
Figure 2
- Linear chromosome map put together
based on the information collected from the Jacob and Wollman experiment. The
marker positions indicate the time of entry of each gene into the recipient
cell. The distances are given in minutes.
A provisory linear map can be
constructed from the different times of entry of each gene (Figure2). Since only one F factor is integrated in each Hfr
strain, this integration appears to occur at random, and the F factor is responsible for the transfer
of the donor cell genes into the recipient cell, different Hfr strains vary with respect to the origin and direction of gene
transfer. So by using different polyauxotrophic Hfr strains, in which the F
factor has become integrated in different sites and in different orientations,
is possible to establish the complete genetic map of the E. coli chromosome. To do this is necessary to measure and compare
the times of transfer of each gene and to identify the overlapping regions. The
simplest way to arrange the genes of the E.
coli chromosome is as a circular one (Figure3).
Figure 3
- Simplified circular genetic map
of E.
coli determined by the interrupted mating experiments
The evidence that the genetic map
of E. coli was circular was very
important because all the previous genetic maps of eucaryotic chromosomes were
designed in a linear way.
From these experiment it was also
possible to determine that the genetic distances between a particular pair of
genes, measured in time units, were constant (within an experimental error
margin), independently of the Hfr
strains used as donors. This finding corroborates the use of time units to
measure the distance between genes in the E.
coli chromosome.
From what we have seen, it is easy
to understand that the use of conjugation and interrupted mating experiments
had a huge impact in genetics, once it allowed to construct a complete genetic
map of the bacterium E. coli. This
map was determined to be 100 minutes long and provides information about the
relative locations of the E. coli
genes in the double stranded circular chromosome. This was the starting point
to the determination of the genomes of higher organisms such as man.
Bibliogarphy
·
Russel, Peter J.,
“Genetics”, 5th Edition, 1998, The Benjamin/Cummings Publishing
Company, Inc, USA