|[Previous] | [Next]|
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
Search | Send us your comments
©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison
III G. MUTANT DETECTION
Detectability of mutations is a function of their effect on the phenotype. All of this discussion of mutagenesis has focused on mutations (i.e. alterations in the primary structure of DNA), but what you typically look for are mutants (cells with altered phenotypes that arise because of those mutations). The likelihood of a given mutation being detected (with or without mutagenesis) is a function of a variety of factors, all of which reflect the fact that one detects only those mutations that display a sufficiently altered phenotype. Other than duplications (see IVA) the most common type of mutation is a base substitution. This is true for both spontaneous mutations as well as those following mutagenesis by a base analog, alkylating mutagen or UV. An inspection of the genetic code (see Figure 3) will show that the majority of these will cause the substitution of one amino acid for another (at least where the product of the mutated region is a protein). The mutations that cause such amino acid changes are termed missense mutations. In the case where you are mutating a wild-type gene, the product of the mutated gene would typically have the same or lowered activity than that of the wild-type, but clearly that activity will not necessarily be zero. It is particularly common for temperature conditional mutants to have significant amounts of activity under the non-permissive condition. The following arguments will suggest the ways in which a partial loss of a product function would affect the phenotype and therefore the detectability of the mutant. (Frameshift, nonsense, and insertion mutations nearly always destroy gene product function so that the following sections which treat partial loss of function do not pertain to them. There is a general summary of the potential effects of various mutations types in section V A.)
"Toughness" of the gene product. If a protein serves a strictly "structural" role in the cell, there may be no one region critical for function. Thus missense mutations will rarely have a sufficiently detrimental effect on function to be detectable. The only mutations that would be seen would be those with drastic consequences for the function of the protein. By a similar line of reasoning, there will always be regions of a product that are more critical than others: a missense mutation affecting the active site will be much more likely to have a discernable phenotype than one that affects a non-critical region of the product and therefore more random base substitution mutations will be detected (as opposed to generated) in regions of the gene encoding critical portions of the gene product.
metabolism of the system. If a wild-type cell produces a protein at a level just barely sufficient for good growth, then any loss of that activity in a mutant would likely be detectable because it would become rate-limiting for the cells growth. On the other hand, a product produced in excess of growth requirements would need to be damaged (in a mutant) much more severely for it to become growth-limiting. As an example, assume there are two steps in arginine biosynthesis (there are more, of course), argA and argB, and that the product of the argA gene has ten times the activity (the number of protein molecules times the specific activity of each protein molecule) of the argB product. A missense mutation in argA that dropped the product activity by 80% would still be undetected since argB product would still be limiting or, more properly, the activity of the argA product has not become sufficiently low to cause a discernable phenotype. A missense mutation in argB with a comparable effect on its product would probably be detected (assuming, of course, this effect was sufficient to make the arginine supply limiting for growth!).
"Regulation" of the system. There are systems of genes that are typically expressed at an intermediate level but can, through appropriate regulation, be expressed at much higher levels if the situation demands (anabolic pathways are often examples of such systems). There are other systems in the cell that, when they are "turned on", are expressed at maximum rate (examples tend to be catabolic systems). "Expression" in these cases refers not only to the amount of RNA transcription of the region (the typical usage), but also to the effects of post-translational regulation of mRNA translatability or stability. Please understand that such regulatory possibilities do not exist because the cell "plans" for mutations, but rather because there certain physiological conditions where elevated expression is useful to the cell.
As an example of regulation affecting the detectability of mutations, E. coli, even growing on minimal medium where it needs to synthesize its own histidine, expresses its his genes at only about 5% of maximal derepression levels. A his mutation that causes a 90% reduction in activity of a his enzyme (even in the limiting step in the pathway) will be undetectable, because the cell is still able to turn up the expression of the mutated gene (in response to its perceived histidine shortage) to a level sufficient to produce enough total enzyme activity (of the mutated gene's product) to make sufficient histidine for growth. In the his regulon of E. coli, therefore, the only mutations detected are those with a drastic affect on the affected gene product. A counter example is the E. coli lac operon, which encodes the gene products involved in lactose utilization. This system is fully expressed when lactose is the best available carbon source and any diminution in activity of the pathway results in slower growth. Not surprisingly, missense mutations are frequently isolated in lac (greater than 95% of detected lac mutations) because they are often detectable while in his they are rather rare (about l0% of detected his mutations).
"Essentiality" of the system. If a gene product is essential (see page 4) for cell growth then a large fraction of mutations in that gene will cause the cell to die and strains bearing such mutations will not be found. The only mutations that will be detected in such genes will be either conditional (e.g. they destroy the essential function only at certain temperatures) or partial loss of function.
Expression of the phenotype. A mutation may not express "its" phenotype immediately, either because:
In the case of a new mutation causing a loss of function, it may take some time for the product of the (pre-mutated) gene to be "diluted out" enough for the phenotype to become apparent, a phenomenon referred to as phenotypic lag. For example, a cell that has just acquired a mutation in hisD (which encodes the final enzymatic step in the biosynthesis of histidine) will still have a significant amount of both functional hisD protein and a pool of histidine which this protein has synthesized. It is only when the amount of functional hisD protein per cell gets too low (because of cell growth and therefore partitioning of the functional hisD protein to more and more progeny cells) to maintain the level of histidine necessary for growth that the genotypic mutant displays its mutant phenotype.
In the case of the new mutation causing an acquisition of function, it may take some time for the product of the newly mutated gene to accumulate, through new synthesis, to a level high enough to display the new phenotype. An example of this might be the reversion of a hisD mutation to a his+ genotype. A His+ phenotype will be detectable only when sufficient functional hisD protein has been accumulated to satisfy the cell's requirement for histidine.
In both examples, the time required will be a function of (i) the level of gene expression before the mutation took place, (ii) the level of gene expression after the mutation took place, (iii) the level of product required for the appropriate phenotype under the conditions used, (iv) the growth rate of the cell, and (v) the instability of the gene product.
Failure to detect a particular mutant type. There are three reasons for not seeing a mutant class: (i) the mutation at the desired gene or site did not occur (you did not alter the genotype appropriately); (ii) you did not recognize the generated mutant because it had an unexpected phenotype; (iii) you did not look hard enough.
Siblings/independence/non-identicalness. If you are going to go to the trouble of biochemically analyzing the mutants you produce in a selection or enrichment, it would be a disappointment to produce and analyze the identical mutant l00 times. How might such a situation occur? Any time your enrichment scheme allows significant periods of growth following mutagenesis or selection, you will tend to get a number of progeny from any cell whose phenotype satisfies the hunt. Such identical progeny are termed "siblings" and by definition they are genotypically identical. This problem can also arise if there are spontaneous mutants, whose phenotype satisfies the particular selection or enrichment, pre-existing in the culture prior to the mutagenic treatment. What can be done about the problem? There are essentially two solutions: The first is to demand independence and the second is to prove non-identicalness. When two mutants are deemed independent it is because they "had to have arisen separately or independently." This does not guarantee that they will be non-identical but it increases the likelihood that they will be and certainly they cannot be siblings. Strains that are non-identical are those that are "shown to be genotypically or phenotypically different from each other". If, for example, you do separate enrichments from each of two single-colony isolates then mutants arising from each enrichment will be independent of each other and will very likely be non-identical. Obviously, if one gets several mutants from the same selection or enrichment (and therefore not independent), but shows them to be non-identical, then you have achieved the desired result. On the other hand, it may well be very time consuming to prove non-identicalness. What you want is genotypically different mutants for further analysis and "independence" provides some assurance of that. However, if you do have a decent genetic or biochemical screen to assay non-identicalness, you may want to analyze a number of non-independent isolates from the same enrichment. If you do not, stick with independent mutants.
[See sample problems 4, 5 and 6]
[Previous] | [Next]