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Summary of mutagenesis
©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison
III F. SUMMARY OF IN VIVO MUTAGENESIS
Mutagens vs. spontaneously derived mutations. If you use spontaneously generated mutants, they may well all be similar due to hotspots (see below) for spontaneous mutations. Worse, they might all be due to strange events like mobile genetic elements (section IV E) or multiple clustered mutations as noted earlier (section III A1). If you rely on chemical mutagens, hot spots are still possible but this is a little less severe than in the case of spontaneous "mutagenesis." The major difficulty with use of mutagens is that you are beating up your organism, possibly generating other mutations that might complicate your life later. Deliberate use of transposons for mutagenesis will be discussed below and can be very useful since they tend to be clean, single hits of a known quality, but they do not generate the most biochemically interesting mutations (those causing altered gene products) in that they are always "knockouts" (complete loss of gene product function) and nearly always polar. In general, you seem to be best off with a light chemical mutagenesis followed by good screens and enrichments. For particular classes of mutation types, you should always spend a little extra time thinking about how to devise a selection (section II C).
Obviously mutagens also increase the frequencies of mutations. Mutagen effectiveness or strength is the result of a complex set of factors: how well the mutagen gets into the cell, how well the various repair systems respond to the particular chemical change stimulated by the mutagen, and how much "killing" occurs relative to the amount of mutagenesis. With these in mind, a generalization can be made that alkylators are very effective (l02-l04-fold stimulation compared to spontaneous frequencies) while the base analog and frameshift mutagens are less effective (l01-l02 stimulation).
The importance of a mutagen's target specificity. Generally speaking, this is not too important a consideration for screens/selections involving "loss of function", since there will be many appropriate sites within any given gene. For example, even a G-specific mutagen will find many targets in any gene; no gene is so low in G's that it is a poor target for a given chemical mutagen. Where a very specific base change is demanded by the selection (typically in a "gain of function" selection), some base substitution mutagens will be better than others depending on the particular base changes they generate. See also the argument on "target size" below.
The fact that frameshifts, whether induced or spontaneous, often occur in redundant runs of bases implies that frameshift mutations will be less random than base substitution mutations. Nonetheless, such short redundant runs of bases occur a large number of times in virtually any gene, so the frequency of frameshift mutations in "loss of function" screens will be a function of the size of the target gene.
There are mutagens (insertion sequences and transposons) that possess much more specificity in their choice of target sequences. If the specificity is sufficiently demanding, some genes will never be mutated by a given mutagen (as in the case of certain transposons, section IV E). The critical question for the experimenter is, what is the range of genetic alterations that cause the desired phenotype and does the mutagen in question cause those mutations at a reasonable frequency.
Importance of "target size" in a gene. Because of the physiology, biochemistry and regulation of the target system, there are a finite number of different genotypic alterations that produce the desired phenotype. The actual frequency at which mutations are seen, with or without mutagenesis, is quite clearly a complex function but several rules apply:
a. There is a much larger target for loss of a product's function than there is for acquisition or alteration of its function. For example, let's say that there are two ways to become resistant to an amino-acid analog, altering the essential target enzyme or eliminating a non-essential permease. The latter will occur 102-103 times more frequently than the former.
b. Due to their effect on the gene product, some mutation types nearly always have a detectable phenotype: deletions, nonsense, frameshift, and insertion mutations. (This of course assumes that the complete loss of the gene product causes a discernible phenotype.) The mutagens that induce mutations exclusively of this type (frameshift mutagens and transposons) thus produce mutations solely on the basis of target size without regard to factors mentioned in the following section.
c. The frequency with which mutant classes occur provides some information about the system that they are affecting. For example, a mutant phenotype which is detected relatively frequently must be arising due to a frequent mutational event like the knockout of a gene product. This line of reasoning will be pursued in the section V.B.
d. Hot spots for point mutations can arise at sites preferred by mutagens, sites where replication/ repair enzymes frequently make errors, or sites that are poorly repaired by repair systems. As described below, large direct repeats can serve as hot spots for duplications and deletions, and certain insertion elements have site-specificity, so hot spots are not restricted to any particular type of mutation. The effect on your analysis will depend on just how frequently mutations occur at these sites, and whether or not they cause a phenotype that you are looking for.
Requirements for DNA replication during mutagenesis. For reproducibility, you want to control the amount of mutagenesis and therefore you want to expose a culture to a mutagen for a set period of time and then remove it from that mutagen. In practice, a culture of bacteria (l08-l09cells) is exposed to a mutagen for some short period of time, the mutagen largely removed by centrifugation or filtering the cells, the cells grown under non-selective conditions for a few generations to allow "fixation" of mutations and expression of mutant phenotypes. The culture then analyzed by selections, screens or enrichments for cells with the desired phenotype.
No mutagen is completely random, there is a wide range of degrees of specificity
When a mutation causing "loss of function" is sought, the number of different mutations yielding that phenotype is probably large and a variety of mutagens (or a variety of different spontaneous events) will be capable of producing such mutations
When "acquisition of function" is sought, the number of particular mutations that cause the appropriate alteration of the product will be small and only those mutagens (or specific spontaneous events) that can induce those particular mutations will be useful.
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