Gene Cloning

146 Gene Cloning
where the transposon jumps from the vector to some position on the
genome, but where no further transposition occurs because the genes for
transposition are lost when the cell divides.
Q6.2. Why is it important to limit the number of transpositions made by
the transposon when attempting to clone genes by transposon tagging?
Second, some method must be available for detecting the novel phenotype.
Since the number of mutants generated overall may be very large, and
the proportion of these which are actually in a gene of interest will be very
small, a key factor in determining the success of this strategy is to have a
very robust method for distinguishing between the mutants of interest and
all the others. Ideally, a selective approach can be used, where only the
mutants of interest survive and all the others die. But other approaches are
also possible, as we will see in the examples below.
Third, although clones in a library with the transposon present can be
identified by DNA hybridization, it is common to use a transposon which
has been modified to make its detection in the library easier. For example,
the selective marker (almost always an antibiotic resistance marker) on the
transposon can also be used to select those clones from the library that
contain the transposon. This requires that the selectable marker is
expressed both in the target organism where the original mutagenesis is
done, and also in the organism (almost always E. coli) in which the library
is screened. Alternatively, some transposons used for mutagenesis have
been engineered to have two selectable markers – one expressed in the target
organism and one in the E. coli host – to get round this potential problem.
An even more useful approach is to put a bacterial origin of replication
onto the transposon as well as a selectable marker. The way in which the
region into which a transposon has jumped can be cloned is illustrated in
Figure 6.3.
Q6.3. Before looking at Figure 6.3, can you see how including a bacterial
origin of replication on a transposon which is being used for gene tagging is
helpful in recovering the region of DNA containing the transposon after tagging
has taken place?
Having used this approach to identify candidate genes, a number of
careful experiments need to be done to confirm that the gene which has
been targeted is indeed the one responsible for the phenotype of interest.
Some indication of this may come from the gene sequence, and the protein
sequence that arises from its translation. Thus if, for example, you are
hunting for a gene producing a protein involved in some aspect of