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Table of Contents
Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
Transposons - class 2 and bacteriophage Mu
CLASS 2 OR COMPLEX TRANSPOSONS (USING Tn3 AS THE EXAMPLE)
Transposition: Transposons of this class tend to show regional specificity and Tn3 in particular seems to especially prefer AT-rich regions that have some homology to its ends. Unlike Class l, the ends cannot transpose separately. One member of this class, Tn7 has such extreme specificity that it has a single site of preference in all of the E. coli chromosome. It has now been shown that the region conferring this specificity is not the site of insertion, but is apparently nearby. A similar separation of recognition and "action" sites has been seen with certain restriction enzymes. The frequency of transposition tends to be variable ranging from 10-4 to 10-6 and, in the case of Tn3, involves a five-base-pair duplication upon insertion.
Regulation of transposition: The structure of Tn3 is indicated in the accompanying figure. What should be noted is that the products of two genes, tnpA and tnpR, are involved in some way in the transposition event. The product of tnpR, the resolvase, is also a regulatory protein, acting as a repressor of both its own synthesis and tnpA, which encodes the transposase. The mode of regulation of other transposons of this class is unknown. Tn3 has also been shown to possess an "immunity" function that reduces the frequency of other Tn3 insertions into the same replicon. The mechanism of this immunity is unknown.
Polarity: The situation with this class of transposons in terms of their polarity is somewhat uncertain. It is quite clear, however, that in one orientation at least, these transposons tend to be polar. It is possible that the lack of polarity in other cases is due to the initiation of new transcripts from transposon promoters and not due to reading through across the transposon.
Deletion generation: Both deletion and inversion events next to the transposon are frequent. The end points of both the deletions and the inversions seem to be non-random and in the case of inversions, there is typically a second copy of the transposon at either end of the inverted region.
Element deletion: Tn3 does not appear to be deleted precisely at a detectable frequency. Even in those few cases where revertants to a wild-type phenotype occur, subsequent analysis has shown that the wild-type genotype has not been restored. Such events that might restore a wild-type phenotype without the wild-type genotype will be discussed in the section on suppressors near the end of the text. For another transposon of this class, Tn101, reversion to a wild-type genotype has been shown to occur at 10-11, which is essentially undetectable.
We will briefly treat a number of the same properties discussed above for bacteriophage Mu (named for its "mutator" effects). For all intents and purposes it belongs in the general category of a Class 2 transposon since it is not flanked by separately transposable insertion elements. Its physical size is 38 kb and it generates 5 base pair duplications upon insertions. It produces an 11 base pair inverted repeat at either end. Its site preference is remarkably random and the argument has been made that its specificity can be for no more than one or two base pairs. However, in at least one particular region, it has been found that a disproportionate number of insertions fall within one very small region of the gene suggesting that there can be some site preference. Mu is rather strongly polar in both orientations, but it is clear that there is an exceedingly low level of transcription out of one end of the prophage. The transposition of Mu is known to generate deletions as roughly l0% of the Mu prophages have adjacent deletions. These deletions tend to start at one end or the other of the prophage and extend into the adjoining DNA though there also seem to be cases where the deletions are unlinked to the prophage. Finally, precise deletion of Mu is rather rare, occurring at approximately 10-9, and seems to be dependent upon at least some Mu factors. The advantages of the use of Mu are: it is not normally found in the bacterial genome and therefore there are few problems with homology to existing sequences in the chromosome; in contrast to most other transposons, Mu does not need a separate vector system (see para E4 below) since it is itself a vector, being a bacteriophage; Mu prophage (at least the cts versions, where c encodes the repressor) are inducible. The disadvantage of Mu is that it is a bacteriophage and therefore can kill the host cell. A wide variety of useful mutants of Mu have been generated.
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