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Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
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Uses of transposons
©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison
IV E4. USE OF TRANSPOSONS
Mutagenesis: Since it is clear that transposons transpose to new sites at certain frequencies, one might expect that you "mutagenize" a strain by taking a strain carrying the transposon (in a site considered "uninteresting" in terms of the system being studied) and looking for cases where the Tn has moved and generated an "interesting" mutation. While this will work, it is not smart: (a) you will not know when a transposition has occurred--both the starting and desired strains have the transposon (i.e. you will not have a selection for the acquisition of the transposon, since it was present to start with); (b) because you will be looking for the loss of gene function (the typical result of transpositions), you will typically not have a selection for the mutant itself. These problems are solved if the transposon is introduced into the cell in such a way that every transposon-containing cell (and therefore, drug-resistant, selectable cell-type) is generated by an independent transposition event. If this is achieved, then you have a positive selection for mutants (cells not mutagenized by the transposon are drug-sensitive) and every survivor of the selection will contain a Tn-induced mutation. Since the frequency of mutagenizing any gene is high (based on target size, about l/3000 for an average-sized gene), most "knockouts" detected in a given gene will be Tn-derived and not spontaneous (this argument assumes a "random" Tn). Such a situation is achieved if the Tn is introduced into a recipient cell on a non-replicating vector. The vector should also not be able to easily integrate itself into a replicon, as would be the case for a specialized phage, see section VI C3. (A vector refers to the DNA, typically a plasmid or phage genome as described in Section VI A, into which the Tn is integrated and which provides the mechanism for getting the Tn into the target cell.) In this case, the only way for a "stably" drug-resistant cell to exist is for the Tn to become associated with a replicon by transposition. This description of replicons and specialized phage will necessarily be obscure until these topics are described in the text but an allusion to them here is necessary in order to describe transposon use.
Selection for a mutant phenotype: Most mutations leading to the loss of gene function do not have a selectable phenotype. This means that they cannot easily (i.e. selectably) be moved from one strain to another using the gene transfer systems described in Section VI of the text. A mutation caused by a transposon therefore has a distinct advantage, since it has two phenotypes: the loss of function of the affected gene and the drug-resistance of the transposon. The latter is selectable, but brings the former along. Note also that a Tn can be viewed as just a selectable marker, allowing linked, but not transposon-mutated, genes to be moved (linkage is defined in Section VIII A).
Cleanliness: As a mode of mutagenesis, transposons are relatively clean; that is, in contrast to other forms of mutagenesis they do not cause a large number of alterations in the genome. While the sections above note the possibility of deletions and inversions caused by insertional events, at worst, only a few percent of the selected insertions are accompanied by such problems. To a first approximation, mutagenesis with a transposon will produce strains with a single insertional event. The downside is they are only knockouts, never alterations of function.
Organism specificity: Because most MGEs borrow some host machinery during transposition, they tend to be limited to hosts with "compatible" machinery. Most MGEs found in Gram-negative bacteria therefore tend to be specific for that group and fail to function in Gram- positives.
Genetically altered transposons: An increasing number of genetically altered transposons are being produced with useful and amusing properties: (1) elements with drug resistance of one transposon but with a transposase and the ends of another, thus yielding, for example, elements with a Tn5 randomness and a variety of drug-resistant properties. (2) Elements that transpose more or less frequently than the originally isolated version due to differential expression of the transposase. (2) Elements that transpose only once and then are stable. These latter types are generated by removing the transposase gene from the element but supplying the gene product in "trans" (see section VII) by placing that gene elsewhere in the donor cell. (5) Elements that carry a gene or operon that is unexpressed due to the lack of a promoter, but expressible if the element is inserted (in the appropriate orientation) into an actively transcribed region. Such elements have been devised so that they do not disrupt transcription entering one end of the element but allow such transcription of the promoterless region they carry. A further discussion of the use of Fusions, as such as hybrid transcripts are called, is found in section X. Such engineered elements typically also carry a drug-resistance gene with its own promoter. As noted in the chapter on Fusions, these elements can be used to screen for insertions based on the way the are regulated, rather than the growth phenotype they cause.
Physically detectable homology: When a transposon is used to generate a mutant, the mutated region can be physically isolated using probes for the transposon sequence. The mutated region can then be used in turn as a probe for the wild-type sequence.
IV E5. TAKE HOME LESSONS WITH INSERTIONS AND TRANSPOSONS
IS and Tn's have a wide range of target requirements and therefore of site specificity.
They nearly always destroy product function of the affected gene.
They tend to be polar but, depending on the element in the site of insertion, they may occasionally provide new transcription of adjacent genes.
The ends of elements are "funny" and stimulate deletions and DNA rearrangements by unknown mechanisms.
Different elements differ in their frequency of precise excision (the restoration of the wild-type genotype).
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