Complex mutations

©2003 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison

IV. MORE COMPLICATED TYPES OF MUTATIONS

IV A. DUPLICATIONS

Duplications are one of the least appreciated and yet most important mechanisms of genetic rearrangements in enteric bacteria and there is no reason to believe they are not common in other bacteria as well as in eukaryotes (duplications are common in bacteria and abundant in my questions!). Duplications are present in E. coli at a very high level: approximately 0.l% of a culture is duplicated for a given region of the chromosome. Because of this high frequency, you may safely assume that any time you have a situation where a duplication will satisfy your selection, that class is virtually all you will be able to detect because they will so predominate. In general, these duplications are rather large, up to one-third of the chromosome, and in a Rec+ background they are highly unstable (approximately l% of a Rec+duplication-containing culture will have spontaneously lost the duplication) as they are typically tandem duplications and are lost by homologous recombination. Tandem duplications are those where the two copies of the duplicated region are immediately adjacent to one another in the same orientation. These duplications tend to be tandem because of their mode of generation which seems to involve either legitimate or illegitimate recombination between daughter strands in the same cell. Since legitimate (homologous) recombination can produce duplications, at least in some regions flanked by homologous sequences, fewer duplications are seen in a Rec- strain. The degree of this reduction varies, but a reduction of l02-fold might be typical. The figure below gives an indication of the sorts of events that would arise if a recombination event occurred between two small regions. While not immediately obvious, to regenerate the two separate daughter chromosomes in a proper topological sense, another recombination event is required elsewhere in the genome.

On the other hand, the absence of a recombination system (Rec-) will stabilize these duplications fairly well ( 10^-6 of a Rec- duplication- containing culture will lose the duplication due to all sorts of illegitimate recombination events). In any duplication there is only one non-wild-type sequence in the entire duplication and that is at the center "join point". For this reason, duplications do not generally cause a discernible phenotype. The only way they cause loss of function is if the duplication is small: internal to a gene, necessarily damaging the gene product; or internal to an operon, where the join point causes a frameshift, leading to polarity onto downstream, non-duplicated genes in the transcript (such small duplications are rare). If the join point causes, for example, the fusion of one gene (see section X and figure 15) to a dissimilar promoter, then this point of fusion within the duplication might well give it a discernible phenotype (but not a loss of function). If the duplication is non-tandem, then homologous recombination between the duplicated regions will cause the loss of the unique material encoded between the duplications. This sort of event is indicated in figure 16. For this reason, a non-tandem duplication will typically be stable since the loss of the unique information between the duplication would typically be severely deleterious for the cell. Perhaps because non-tandem duplications require two non-homologous recombination events for their generation, their occurrence appears to be very rare compared to that of tandem duplications. There are several cases in E. coli of non-tandem duplications, notably the argI and argF genes as well as tufA and tufB, and at least one of these seems to have been caused by transposition of the second copy, rather than an error of homologous recombination. [See sample problem 7]

IV B. AMPLIFICATIONS

An allied subject is amplification, which might be defined as duplication run amuck. Specifically, it has been shown for a variety of Streptomycetes (at least) that after various abusive treatments, one finds massive tandem duplications of small regions of the chromosome. While it is not clear at this point what the function of these is, it does make it clear that a system for generating such an amplified region does exist in at least some prokaryotes.

IV C. INVERSIONS

Inversions are the sorts of events that would be found if homologous recombination occurred between two regions of the chromosome whose homology occurred in opposite directions on the DNA. While this description helps to picture such an event, note that inversions are not necessarily caused by homologous recombination. Unlike tandem duplications, inversions have two non-wild-type join points. These inversions are harder to detect than duplications (since they lack the characteristic instability of a tandem duplication) and therefore have not been the subject of much analysis. An obvious example of an inversion is the trp-cysB region (approximately l0% of the chromosome) of E. coli, which is inverted relative to that in Salmonella typhimurium.

There are a number of cases in the literature where very specific regions undergo inversion as a regulated, not a mutagenic event. In these cases, a unique, site-specific recombinase mediates the reversible inversions. In these cases, different gene products are produced depending on the orientation of the region. Examples include gin of bacteriophage Mu and hin of S. typhimurium.

IV D. DELETIONS

Deletions may be defined as the loss of three or more bases relative to the wild-type sequence (clearly the distinction between frameshifts and small deletions is arbitrary). They are detected with fairly high frequency (10-6 might be typical, but this number is very dependent on the region of the chromosome examined. It should be remembered that the observed frequency of deletions, and any other type of mutation, will be a function of the selection employed. The particular selection noted demanded very tight mutations and thus a reasonable percentage of detected mutations were deletions.) Spontaneous deletions seem to be generated with at least some regard to small regions of homology and, as will be mentioned below, deletions are often stimulated by the presence of insertion sequences. As with duplications, the fraction of deletions which occur due to the recA system is variable depending on the region analyzed, but tends to be a significant fraction. In one well-studied case, deletion formation was reduced twenty-fold in a Rec- background (reminiscent of the Rec- effect on duplication formation!). Deletions can also generate either transcript or protein fusions (see section X), where non-identical transcripts or genes are fused together respectively. Finally, deletions can be of almost any size, with the constraint that your strain will be dead if you delete an essential function. These facts should be reminiscent of those for duplications and the two processes may reflect the two products of recombination based on short sections of homology (figure 11). Deletions may well occur at the same frequency as duplications, but are detected less often because they will often be lethal events.

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