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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
III. GENERATION OF POINT MUTATIONS
Obviously, for the isolation of interesting mutants for eventual biochemical characterization, one needs to generate such mutants at a frequency in the population which allows their detection. The previous section on genetic methods has provided some indications of the power of various screens and selections. The following sections treat the frequency of mutant generation and factors involved in their detectability.
III A. INHERENT MUTATION FREQUENCY
Understanding the actual mechanism by which rare spontaneous mutations occur has been an exceedingly difficult research problem and will not be discussed here. Suffice to say that, through some combination of errors in DNA synthesis and occasional failures in DNA repair systems (see section IIIC), the frequency of any base being mutated to any other is about 10-7-10-8. Therefore, if you started with a mutant strain containing a base substitution mutation, you might expect it to revert to the wild-type genotype at about this frequency. However, the measured reversion frequency (which is the return to an apparently wild-type phenotype) will typically be higher than this. This is the case because often a variety of base changes (starting with the mutant genotype) will restore a wild-type phenotype (or at least one close enough to the wild-type to satisfy the selection) and yet will not restore the wild-type genotype. Some geneticists, even some of my friends, use the term "pseudo-reversion" to refer to those revertants that do not restore the wild-type genotype. I find this usage awkward, because you would not know whether to call "it" a "revertant" or a "pseudo-revertant" until you sequence it. The 108 figure thus provides a kind of benchmark or base line for interpreting reversion frequencies.
For our purposes, "order of magnitude" estimates are satisfactory so the following simple methodology will suffice. Operationally, one grows up a full density culture of the strain under examination and plates out about 108 cells under selective conditions. If, after an appropriate period of growth (perhaps 1-3 days for a typical bacterium), one sees 102 colonies, the mutation frequency for the selected phenotype is said to be 10-6 (102 colonies/108 cells plated). This method may seem to have little connection with the first calculation which deals with "base pairs per generation". However, when one plates out 108 cells, one is effectively examining the mutation frequency of a region which has undergone 108 replications so that the number of mutants seen reflects the frequency of mutations capable of causing the selected phenotype after 108 replications. If the only mutation capable of satisfying the selection is a return to the wild-type genotype, the frequency of revertants will be about 108 in the case of a base-substitution mutation. In practice, mutation frequencies determined in this way will be a little high, since some small amount of growth will often occur on the selection plate by all the plated cells. The assumption of "108 plated" cells will therefore be an underestimate by a factor which reflects the amount of growth that occurred. For our purposes, we will ignore these complications and assume that the experimentally derived number is correct. We will use the term "spontaneous", as in "spontaneous mutation frequency", to indicate "the lack of mutagenic treatment by the experimenter".
In the discussions that follow, we will often use the term point mutation. This refers to any mutation (e.g., base substitution or frameshift, see section III E) where only a single base of the DNA is affected in the mutant when compared to wild-type. Typically such mutations revert at easily detectable frequencies (greater than 10-8), in contrast to deletion mutations which affect a number of bases and can almost never be restored to a wild-type phenotype at a detectable frequency. The term marker will also be used to refer to a mutation (of any type) which has a scorable or selectable phenotype (and hence can "mark" a region of the chromosome).
The above section clearly implies that base substitution mutations are an expected type of spontaneous mutations and later sections will discuss deletion, frameshifts, duplications ,and inversions, all of which occur spontaneously at detectable frequency. It has very recently become apparent, however, that a surprisingly common type of spontaneous mutation is actually a cluster of two or more lesions (including base substitutions and frameshifts) within a few base pairs of each other. The mechanism by which these are generated is not completely clear, but there are suggestions that they sometimes result from single events where a nearby similar, but not identical, region is used as a "template". If these complicated mutations are shown to be a substantial fraction of spontaneous DNA lesions, it would be another reason to utilize mutagens for the production of mutants.
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