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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
IV E. MOBILE GENETIC ELEMENTS
Mobile genetic elements (MGE) are of interest for the insight they provide into basic molecular biology and evolution, as well as for their use as basic genetic tools. They are becoming increasingly difficult to discuss because they are so varied in their properties that they defy consistent nomenclature. For example, some have defined MGEs as regions of DNA that insert into new sites by a "transposition" mechanism, which involves the synthesis of at least a bit of DNA (see IVE3). It has become apparent, however, that some elements move by a mechanism similar to that used by phage lambda (see VIC3), involving site-specific recombination, but no DNA synthesis. In all other respects this last class "acts" like other MGEs. Whether or not they are MGEs has therefore become a semantic problem, depending largely on the definition chosen. There is little doubt that all currently described "transposition" mechanisms involve some form of Rec-independent, or non-homologous recombination. In terms of their use in genetics, transposons carrying drug-resistance genes provide selectable, "clean" mutagens. As mutagens, they destroy the function of the product of the mutated gene, tend to be polar, and have site specificity ranging from very specific to nearly random. The point of the following examples and sections is not to give specific information but to give a rough idea of the range of possibilities which have been described so far.
IV E1. INSERTION SEQUENCES
Insertion sequences (IS) are defined as mobile genetic elements that are known to encode only functions involved in insertion events. This is to be contrasted with transposons (Tn) that are mobile genetic elements containing additional detectable genes in addition and unrelated to insertion functions (for example, drug resistance). Since IS elements, by definition, do not carry other genes, they tend not to be used as genetic tools. On the other hand, mutations caused by IS's tend to be fairly common among spontaneously derived mutants, so that knowledge of their properties is relevant. Finally, recognize how very arbitrary the above definition of an IS is: it is potentially our ignorance of other encoded products that so identifies it as an IS instead of a Tn.
In either case, MGEs are essentially pathogenic elements whose environment is the genome of the host. As in the case of any pathogen, they can neither be too virulent (where they efficiently kill the very host on which they depend for their existence) or not virulent enough (in which case they cease to be identifiable as pathogens). The virulence of these pathogens depends on their "growth" (enrichment in the genome) as well as on any negative effects caused by the insertion itself. The former is a function of the frequency of transposition, while the latter reflects their target sites. Not surprisingly, then, MGEs spend most of their regulatory efforts trying to transpose as infrequently as possible to avoid putting their host at a selective disadvantage. Some elements also try to aid the host by supplying useful functions like drug-resistance.
The following is a "laundry list" of properties to provide a range of the possibilities:
Physical properties. The majority of IS elements are between 0.7 and l.8 kb in size and the termini tend to be l0 to 40 base pairs in length with perfect or nearly perfect repeats. These sequences also tend to have RNA termination signals as well as nonsense codons in all three reading frames and are therefore polar. Typically they encode one large open reading frame of 300 to 400 amino acids and by definition the protein encoded by this reading frame is involved in the transposition event. Two exceptions to the size range given above should be noted: The first, }; is 5.7 kb and the other, IS101, is a scant 0.2 kb in size. Although there are exceptions, insertion sequences tend to be present in a small number of copies in the genome. For example, IS1 is present in 6 to l0 copies in E. coli chromosome while IS2 and 3 are typically present in about five copies.
Transposition: Transposition is often referred to as "hopping" and this term has the strong connotation that the element leaves one site and goes to a new site, restoring the original site. This restoration is probably one of the few things that do not happen in transposition. Even the idea of "leaving one site" only seems to be true for one of at least two types of transposition mechanism, the so-called conservative mode of transposition. This is to be contrasted with the replicative mode of transposition that involves the generation of a second copy without alteration of the initial copy of the transposition sequence. These two mechanisms will be discussed in section IV E3, but they share the property that neither restores the original site to its wild-type sequence.
Deletogenic properties/DNA rearrangement. Insertion sequences mediate a variety of DNA rearrangements. One of the first recognitions of this fact was the involvement of insertion sequences in the integration of F and R plasmids into the host chromosome. This event gives rise to Hfr strains and will be discussed in the section on conjugation. The initial DNA rearrangement mediated by IS elements is the "insertional duplication" that they tend to generate at the site of insertion. IS1 generates an 8 or 9 base pair duplication while IS2 generates a 5 base pair duplication. The modes of generation of these duplications will become obvious when the actual mechanism of transposition is discussed below (see IVE3). As noted above, several elements cause no duplication at all, and integrate into target DNA by a completely different mechanism which is related to the site-specific system employed by many phage.
Less common but in some ways more important to the genetic use of insertion sequences and transposons is their deletogenic properties. Different insertion sequences are known to cause deletions in neighboring genes and occasionally delete themselves at reasonable frequencies. For example, IS1 causes deletions in neighboring genes at a frequency of 10-3-10-4 per generation and the insertion itself remains intact. These deletions tend to start at the IS1 element with the other end of the deletion being reasonably non-random and possibly involving some sort of short section of homology. It has recently been shown that the position of the IS relative to promoters in the region affects deletion formation, by unknown mechanisms. IS2 is similar but its ability to generate deletions is rather less. Finally, IS4 makes deletions of both itself and DNA on both sides with fair frequency. Apparently then, the theme of causing the deletion of neighboring genes is fairly common to insertion sequences. Most, but not all, mutations caused by IS's revert (i.e. restore the wild-type phenotype, often by restoration of the wild-type genotype) at detectable frequencies. In many cases, reversion involves the precise deletion of the IS (including one copy of the duplication which was generated upon insertion), restoring not only the wild-type phenotype but also the wild-type genotype. In some literature, this is referred to as "precise excision" but I have chosen not to use this term since it connotes more about the mechanism than is known. This deletion event does not seem to be Rec-dependent and does not even seem to be dependent upon the transposase function encoded by the insertions. On the other hand, this last observation was made in the case of Tn5 and it is possible that with other insertion sequences, the transposase would be essential for precise deletion. Another form of reversion has been observed with a gal::IS1 mutation, where a second IS1 has transposed into the region, restoring downstream gene expression (see below).
By mechanisms that are unclear, many insertion sequences can stimulate an increase in the level of both duplications and inversions. In the case of duplications, multiple copies of the same IS could obviously be used by homologous recombination to generate duplications as shown in the accompanying example. Some insertion sequences, specifically IS5, seem to be able to stimulate Rec-dependent recombination within 50 to l00 base pairs of the insertion sequence.
Finally, if a region is bounded by two identical IS sequences, the entire region can transpose. Such is the case with one of the two classes of transposons which will be discussed shortly. The take-home lesson in all these stories is that transposons can mediate a number of amazing events and it remains to be seen what "rules" will finally emerge.
Promoters/terminators. Typically IS's are strongly polar onto downstream genes because they contain both RNA and protein termination signals. Occasionally, particular insertion mutations are found (IS1, 2, 3, and 5 mutations have been so described) that allow significant expression of downstream genes presumably because the transposon is supplying a new promoter for those genes. The reason for the inconsistency in the occurrence of such "restarts" might have several causes: (i) the potential promoter may exist only at one end of the element; (ii) the potential promoter may be incomplete, relying on the sequence into which the element is inserted for occasional "completion" (IS1 has now been shown to supply a "-35" promoter region and its preference for AT-rich sequences often places it near sequences that can function as "-10" regions.); (iii) the transcripts made from these promoters may not possess a ribosome binding site so that Rho-dependent polarity affecting this transcript is very strong unless the element is inserted immediately adjacent to a ribosome binding site.
Polarity typically refers to a decrease in expression of downstream genes and it is functionally difficult to tell whether this lowered transcription is actually transcription starting at the normal promoter and reading through an element or if the transcription of the downstream gene is entirely due to a new promoter in the insertion sequence itself. This has important implications in trying to understand the regulation of the system under study since only the former case would suggest that the remaining transcription was under normal regulation.
Target specificity. If there is to be a general rule with IS's in terms of their target specificity, it is that they prefer A/T-rich regions in the DNA. Some elements, like IS50, seem to have no greater requirement than that. Others, an example is IS4, have extreme specificities demanding in this case the sequence AACN2-3 CANll-l9 GTTT (where "N" is any base), which exists in only 2 to 4 sites in E. coli. In another case, a section of DNA has been identified that enhances IS2transposition into or near the region in question and in an orientation-specific fashion. As with other questions involving IS sequences, the essential truth is that there are no truths: some insertion sequences are very specific in their target specificity and others are extremely general.
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