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6 BACTERIAL AND PHAGE GENETICS
8 Transposons provide a useful method of mutagenesis
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- A transposon (transposable element)
is a DNA sequence able to insert itself (or a copy of itself) at a new location
in the genome, without having any sequence relationship with the target locus.
- A transposase is the enzyme activity involved
in insertion of transposon at a new site.
- An insertion sequence (IS) is
a small bacterial transposon that carries only the genes needed for its own transposition.
- A selectable marker is a gene that gives a cell
properties, such as resistance to an antibiotic, that allows the isolation of the
cell in a genetic selection.
- A vector is a plasmid or phage chromosome that
is used to perpetuate a cloned DNA segment.
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- Transposons are DNA elements that
can move from place to place in the genome.
- Because transposons carry unique
sequences and frequently carry selectable markers, their location in the chromosome
can usually be easily determined.
- When transposons insert in the chromosome,
they disrupt the activity of the gene into which they have inserted.
- Some transposons integrate almost
randomly.
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Figure 6.10
A deleted version of Tn10 containing only the essential sequences for transposition
and a selectable marker, tet R, is used for mutagenesis. The
miniTn10 is incorporated into the DNA of a vector such as bacteriophage lambda,
which also encodes the transposase. The transposase, expressed from the lambda genome,
cuts the mini-Tn10 out of the chromosome and integrates it randomly into the host
chromosome. This lambda derivative is mutant; it cannot replicate its DNA or lysogenize
the host; therefore, it is lost soon after infection. Each tetracycline-resistant
cell will have the miniTn10 inserted at a different position in the chromosome.
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Transposons, shown in Figure 6.10, are naturally occurring genetic
elements that have the capacity to move — jump — from one location to
another. Sites at their ends are recognized by a specific enzyme or enzymes (transposases)
that are capable of carrying out the cleavage and ligation activities that are necessary
to insert them into a novel position elsewhere on the DNA. The enzymes that carry
out these activities have some similarity to the enzymes involved in retroviral
DNA insertion into eukaryotic genomes. Insertion sequences (IS)
are simple transposons found in bacteria that only encode insertion functions. More
complex transposons may also carry genes coding for other functions.
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In bacterial plasmids, transposons often include genes encoding antibiotic resistance.
Thus, both transfer of the plasmid from cell to cell and the movement of transposons
from one piece of DNA to another can spread the resistance genes, particularly when
a strong genetic selection is present (for instance, when animals or humans ingest
antibiotics).
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When transposons are used in a mutagenesis, as shown in Figure 6.10, the antibiotic
resistance genes are highly useful as selectable markers in tracking
the transposon. A transposon carrying an antibiotic resistance gene is introduced
into the cell on a vector, such as the λ delivery vector, that has been engineered
so that it cannot persist in the cell; therefore, any resulting antibiotic-resistant
colonies have the transposon integrated somewhere in their chromosome. One can select
for antibiotic resistance and also screen or select for the mutation desired. Because
a large amount of DNA is being inserted into a gene, mutagenesis with an insertion
element will usually disrupt gene function.
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Bacteriophage Mu is both a phage and a transposon, named Mu because of the high
frequency of mutations associated with its growth in cells. The mutations are a
direct consequence of part of its life cycle — integrating randomly in the
bacterial DNA as part of both its lysogenic and lytic replication cycle.
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The frequency of transposition of most transposons is low; there are usually hundreds
rather than millions of events, all over the chromosome. Identifying the one that
gives the desired phenotype may be difficult. In addition, transposons do not always
integrate randomly into the genome and the degree of specificity varies with the
transposon. Mutations in the transposase can increase the frequency of transposition
and help randomize insertions (4204). Furthermore, transposition can also
be carried out in vitro at high efficiency into a target piece of DNA,
and the mutagenized DNA then inserted back into the cell (4218).
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Thus far, we have discussed the various kinds of ways to disrupt gene activity,
using mutagens and transposons. Once we have made mutants, how do we detect them?
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Last Revised on December 15, 2003
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- 4204 Kleckner, N., Bender, J., and Gottesman,
S. (1991). Uses of transposons with emphasis on Tn10. Methods Enzymol. 204,
139-180. PubMed
- 4218 Hamer, L., DeZwaan, T. M., Montenegro-Chamorro,
M. V., Frank, S. A., and Hamer, J. E. (2001). Recent advances in large-scale
transposon mutagenesis. Curr. Opin. Chem. Biol. 5, 67-73. PubMed Journal
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©Jones and Bartlett Publishers (2007)
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