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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 1
IV E2. TRANSPOSONS
As defined above, a transposon is a mobile genetic element containing additional genes unrelated to transposition functions. In general, there are known to be two general classes: Class l or "compound Tns" encode drug resistance genes flanked by copies of an IS in a direct or indirect repeat. A direct repeat exists when the two sequences at either end are oriented in the same direction while an indirect (or inverted) repeat exists when they are in opposite directions. In this class of transposons, the IS sequence supplies the transposition function. These are essentially IS's, wherein two copies of the IS have flanked another gene. Examples are Tn5, 9, 10, 903, and 1681. In all of these, the ends of the transposon are capable of transposing separately, since they are in fact still IS's. The second class of transposons are known as "complex" or Class 2. With these, the element is flanked by short (30-40 bp) indirect repeats with the genes for drug resistance and transposition encoded in the middle (see figure of Tn3 below). Not surprisingly, these must always transpose as a unit. Examples are Tn1, 3, 4, 7, 501, and 551. The following sections will consider five general properties of transposons, namely transposition, its regulation, polarity, deletion generation, and precise excision. These properties will be treated first for Tn10, which is an example of the Class l transposons, and then with Tn3, an example of Class 2 transposons.
CLASS 1 TRANSPOSONS (SPECIFICALLY Tn10 WHICH IS FLANKED BY TWO COPIES OF IS10)
Transposition: Transposition of IS10 or Tn10 occurs at around 10-7 per element per generation. Transposition occurs to a large number of sites in E. coli but excellent target sites are found at about 1 per 1000 base pairs. Other, secondary, sites are recognized at poor efficiency. The preferred sites for IS10 and Tn10 tend to have the sequence GCTNAGC (admittedly not very AT-rich) which is then duplicated within the 9 base pair duplication upon insertion.
Regulation of transposition: Kleckner has found that transposition is regulated by a large set of factors and circumstances. These all serve to reduce the level of transposition: (a) As shown in the figure, there are two RNA's produced from one end of the IS10, one which encodes the transposase and the other which is non-coding. These two promoters are separated by 33 base pairs and when the RNA's are both being synthesized and can hybridize to each other, the presence of the second RNA inhibits the translation of the transposase encoded by the first. This second RNA is therefore a trans-acting inhibitor of transposase expression. The Pout promoter is also much stronger than Pin. (b) The coding mRNA appears to be relatively unstable and poorly translated, even without this complementary RNA. (c) Transposase is essentially cis-acting, so it does not float around to cause transposition of other copies of the transposon. (d) Transposase has a dramatic preference for hemi-methylated (due to the dam system) ends of the transposon, resulting in a 104-fold increase in transposition following replication through the element. This restricts transposition both to a very short time frame and to a period where there are at least two replicons in the cell. (e) The transposase functions as a multimer, so single proteins are ineffective at causing transposition.
Polarity: Tn10 is polar on downstream genes, but can turn on downstream genes if and only if the strain is deficient in the function Rho or if all Rho-dependent sites are removed between the site of the transposon and the gene in question. The promoter that allows the activation of these downstream genes is Pout.
Chromosomal deletions: With Tn10, it is possible to demand a loss of the material between the flanking IS10 elements because a selection exists for tetracycline sensitivity (Tn10 confers tetracycline resistance). A variety of events are detected but typically one finds deletions which start at the IS10 and delete all the intervening information between that and the other IS10. Such events are often accompanied by inversions of contiguous DNA sequences beginning at one of the IS10 ends.
Precise deletion: Precise deletion of IS10 or Tn10 occurs at approximately 10-8 per element per generation. This seems to be independent of the recA system and may reflect a "copy-choice" mechanism in replication. Another event, termed "nearly precise", causes the deletion of all but 50 base pairs near the ends of the IS elements. These events occur at around 10-6. Surprisingly, at the frequency of 10-6, these imprecise deletions can give rise to a precise deletion event (a return to the wild-type genotype!).
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