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Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
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©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison
VII A. DEFINITION
A complementation analysis asks if two putative alleles1, when in the same cell2 and acting independently3, can supply all functions necessary4 for a wild-type phenotype5. Complementation is therefore a test of function. The superscripts in this definition are explained below:
The "two putative alleles" refers to two versions of the same region of the chromosome, each of which separately confer a mutant phenotype. They are termed "putative" alleles since it is their very "allelism" which will be determined in this test (they are allelic if they are in the same complementation group). Each allele ought to be present in a single copy number in the cell and it is crucial that the entire relevant region be present in diploid in the cell. Such a partially diploid cell is termed merodiploid. An inverse genetics application is "cloning by complementation", but this will have many of the same concerns as standard complementation with the added concern of copy effects if multi-copy plasmids are used.
The two alleles can either be present on the chromosome or on extra-chromosomal elements. If either version is in more than one copy, there can be both regulatory complications (e.g. titration of a regulatory factor) and difficulties in interpretation (e.g. you do not know if a positive result is due to inappropriate quantities of the product encoded by the multi-copy gene).
Care must be taken that the mutations cannot recombine to form a wild-type genotype so that typically Rec- strains are used.
Only functions absolutely necessary for the desired phenotype, under the conditions used, are "demanded" by a complementation test. Mutations affecting genes whose products are not essential for the desired phenotype will not be tested for in complementation analysis.
The ""wild-type" phenotype" demanded by this analysis should be more rigorously called an "apparently wild-type phenotype under the conditions used". The phenotype is typically scored as "growth" or "no growth", but biochemical assays of the encoded gene product can be performed for more precise quantitation.
Figure 29 gives an idea of the results one could expect from straightforward complementation tests. In these examples when the two mutations in the separate mutant alleles affect the same gene, then neither is capable of generating a wild-type product of that gene and the resultant merodiploid strain is mutant in phenotype. On the other hand, if the two mutations affect different genes, so that each copy of the region is able to generate some of the gene products required (and between them all necessary gene products are synthesized) then the resulting strain is phenotypically wild-type. One problem with this set of examples is that no one(in doing bacterial genetics) routinely puts the two alleles in the "cis"-configuration as a control for complementation (you do build such strains for other purposes, however). It is too hard (for reasons we will cover when we get to Mapping) and it provides very little information, since the presence of the wild-type allele on the other copy will nearly always be dominant. It is, however, often appropriate to consider effects of a mutation on genes in cis, but this is not the same as generating "double mutants" affected in the same small region.
There are three sorts of controls useful in analyzing the results of complementation experiments (see Fig. 30): (a) If either copy of the merodiploid contains a wild-type region, the phenotype of the resulting strain should be a wild-type phenotype, and the wild type is said to be dominant to the mutant. If it is not, the mutant allele is said to be trans-dominant to the wild-type (see section VII B). In either case the merodiploid has the phenotype of whichever allele is dominant. (b) A merodiploid strain constructed with the same mutant allele in each copy should display the mutant phenotype. If it does not, it suggests that mere diploidy for the region of interest can confer a wild-type phenotype. One way of this occurring would be if the mutation conferred a leaky phenotype so that a double dose might yield a pseudo wild-type response. (c) The result should not depend on the location of the alleles; i.e. the same result should obtain no matter which allele is on the chromosome. If this is not true, it indicates that the two locations are not equivalent and therefore the test has marginal validity. This is a variation on the concerns noted for multi-copy plasmids above.
VII B. COMPLICATIONS IN COMPLEMENTATION ANALYSIS
The above examples would seem to suggest that if two mutations complement each other, then they must affect different genes and gene products. This would suggest that the results of complementation analysis would be to define the number of genes in the region. In fact, what complementation analysis does is to define the number of cistrons or complementation groups. More often than not, the number of cistrons will be coincident with the number of genes, but there are a number of special cases where this correlation will not hold. The complications that give rise to these special cases are discussed below and they fall into two general classes: when the non-complementing mutations actually do map to separate complementation groups (paragraphs 1 and 2 below), and when complementing mutations actually map to the same complementation group (paragraph 3 below). Examples of the first class will be detected when the appropriate controls are done, as described above. The second class will be seen as an anomaly in the actual complementation results.
Cis-dominant mutations are a reasonably common type of complication in complementation analyses. Cis-dominant mutations are those that affect the expression of genes encoded on the same piece of DNA (as the mutation itself), typically transcriptionally downstream, regardless of the nature of the trans copy. Such mutations exert their effect, not because of altered products they encode, but because of a physical blockage or inhibition of RNA transcription. There are two dissimilar examples of these sorts of mutations: (a) If a mutation in a transcriptionally upstream gene exhibits strong polarity onto downstream genes, then that mutation has the property of eliminating more than one gene product function. (b) Similarly, a mutation in the promoter or in other regulatory regions outside the translated area, may well eliminate transcription of the entire operon and thus be negative in complementation for all gene functions encoded by that operon. In each of these cases, the mutation is eliminating the function of genes that are themselves genotypically wild type. The mutations are said to be cis-dominant because the expression of the genes downstream on the same piece of DNA will be turned off regardless of the genotype present in the trans copy.
Negative complementation. Another complication involves the very rare phenomenon known as negative complementation or trans-dominant mutations with mutant phenotypes. Mutations of this type cause the resultant merodiploid strain to have a mutant phenotype even when the other copy of the region is genotypically wild type. The phenotype of the mutant allele is thus trans-dominant to the wild type (obviously the reason that wild type is dominant to most mutants is because it supplies the function that they have lost by mutation). There are three general schemes that can be envisaged for mutations causing this sort of phenotype. In each of them, it is necessary to propose that the mutant allele generates a product that, while not wild type, nevertheless possesses some activity that leads to the mutant phenotype. Possibilities include (a) multimeric enzymes where the merodiploid strain would generate multimers whose subunits come from both the mutant and wild-type genes in a random assortment. As shown in figure 31, if the protein was a tetramer, and if any multimer containing one or more mutant subunits was completely inactive, then the presence of the mutant chain would decrease the amount of functional wild-type gene product by approximately 8-fold (this number ignores regulation and assumes a two-fold dosage of the product due to a two-fold dosage of the gene). (b) The mutant gene might cause the generation of an altered protein that interfered in some reaction with the cell and thus caused a deleterious phenotype. In this case, the presence of a wild-type allele would restore the function missing in the mutant but would not eliminate the deleterious phenotype caused by the mutant protein. Thus, the mutant phenotype would be dominant to wild type. (c) It is also conceivable that the mutant copy generates an altered protein that, while it could not carry out the wild-type function, might be competitive with the wild-type gene product. In each case, an altered product is responsible for the trans dominance. Remember, these are rare, special cases: in general, the wild-type allele is dominant to the mutant since the latter typically involves loss of function which is "replaced" by the product of the wild-type gene. Such trans-dominant mutants are very appropriate for further biochemical analysis because the protein product has alteredfunction, rather than merely a lack of function.
Intragenic complementation is yet another possible complication in complementation analysis. This term refers to cases where two mutations that do affect the same gene, and therefore the same gene product, are able nonetheless to give a wild-type phenotype in a complementation analysis. There are two general cases of such a phenomenon: (a) If the product of the gene in question is a bi-functional protein, especially when those functions are independent of one another, then the gene itself will often show intragenic complementation. Such an example is easiest to understand if the product is pictured as "two beads on a string". If each "bead" had an independent enzymatic function, one could imagine that a mutation affecting either (but not both) of the two functions might well leave the other function intact. If two such mutations were put in a merodiploid situation, each would be able to produce one of the two required enzymatic functions, giving rise to a wild-type phenotype. In the case of such a gene, intragenic complementation would be fairly common such that many mutations would affect only one of the two functional regions. This model also predicts that mutations affecting each of the two functions would cluster at either end of the gene creating two clear complementation groups. (b) It is also possible, though less likely, for pairs of complementing mutants to occur in cases where the gene product is a multimeric protein. In such cases, a particular mutant allele might give rise to a protein product that can only function when allowed to aggregate with another particular mutant allele. Such an example is sketched below. In this case, unlike the case of bifunctional protein above, instances of intragenic complementation will be rare, limited to specific pairs of mutants. Further, there is no a priorireason to predict any clustering of complementing or noncomplementing mutations. Would such a case, where two mutations out of 100 in a given gene are capable of complementation, be sufficient to say the gene had two complementation groups? This question is largely a semantic one, but in general, unless intragenic complementation is fairly common, the few exceptional complementing pairs would not be said to define separate complementation groups.
"Unimportant" genes. Since complementation analysis treats only those functions necessaryto generate the required phenotype, it does not allow the detection of complementation groups unless their products are required for the phenotype in question. If, for example, a region encoding such an "unimportant" product (at least under the conditions of the selection) is transcriptionally polar onto an "important" function, that pair of genes has the complementation properties of a single complementation group. This reflects the fact that the only mutations detected in the transcriptionally upstream gene would be ones polar onto the functionally important gene downstream.
The example described in sample problem 15 illustrates some of the expected results from a complementation analysis of a more complicated region. In this example, transposons have been assumed to be polar and point mutations to be non-polar. These assumptions are not unreasonable but, as the section on transposons describes, there can be transcription emanating from the element so that occasionally some expression of "downstream" genes is detected. If that transcription was high enough, the element might appear to be non-polar in a complementation assay. Similarly, some point mutations (frameshifts and nonsense mutations) display at least some polarity and, (as always) depending on the amount of expression of the downstream genes necessary to provide a wild-type phenotype, will be negative in complementation for downstream function.
[See sample problems 15-19]
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