Genetic Techniques for Biological Research Corinne A. Michels Copyright q 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)

5 Complementation Analysis: How Many Genes are Involved?

Complementation analysis is used to determine whether two independent mutations arealterations in the same gene; that is, they are alleles, orare alterations in different genes. In essence, a complementation analysis is a functional test used to define a gene. If a researcher has isolated anumber of mutants with a similar phenotype, the next question asked is: ‘How many genes have I identified?’. If there are 10 mutant strains, are they each in different genes, does each mutant carry a different mutation (allele) in the same gene, or something in between such as two genes one with six alleles and the otherwith four alleles? Complementation analysis will help answer this question. Seymour Benzer’s study of the rZZ locus of phage T4 is a most elegant example of the power of complementation analysis (Benzer, 1955). Benzer had several hundred mutations that gave the same phenotype, large plaques on one host strain of E. coli and no plaques on another host strain, and mapped to the same region of the T4 chromosome. Using the host strain in which rll mutants formed no plaques, he found that when host cells were coinfected with different mutant pairs some pairs produced a normal phage burst while others did not. Those that produced a normal burst he concluded were in different functional genetic units that he called cistrons, a term that is synonymous with gene. In contrast, those pairsof mutants that rarely or never produced a normal burst he concluded were in the same cistron. The rare productive infections Benzer proposed resulted fromrecombination between the different mutations in the same cistron thereby creating a wild-type genotype in a few coinfected cells. Using this method, he placed all of his rll mutations into two cistrons that he called rllA and rllB. Moreover, he made a detailed genetic map based on recombination frequency between the different mutations (a fine structure map) that also indicated the frequency at which a mutation was isolated at that position. In this way he demonstrated that genes are not indivisible units but consist of many mutable sites that can recombine. Geneticists working with other organisms soon followed Benzer’s lead and adapted complementation analysis to their systems. To carry out a complementation analysis, both mutant genes must be expressed in the same cell so that their gene products are synthesized in the same cytoplasm and can functionally interact. Only loss of function (recessive) mutations can be used for a complementation analysis. The theory behind the complementation analysis is simple. If both mutations are loss of function alterations of the same gene, then the diploid cell carrying these two mutant genes will not contain a functional allele and will have the mutant phenotype. If both mutations are loss of function mutations but in different genes, then the diploid cellwill have one mutant allele and one functional wild-type allele of each gene and, since the mutant alleles are loss of function alleles, the diploid will have the wild-type phenotype. 74 GENETICTECHNIQUESBIOLOGICAL FOR RESEARCH To express both mutant genes in the same cytoplasm a heterozygous diploid must be constructed. The way the researcher establishes the diploid state varies with the organism under study. In Saccharomyces, this is accomplished by mating a MATa strain containing mutation #l to a MATa strain containing mutation #2. The ala diploid will be heterozygous for the mutant genes. The phenotype of the hetero- zygous diploid is then observed. If the diploid has a wild-type phenotype, then the mutations are said to complement and this is strong evidence that the mutations are in different genes. A geneticist might also say, ‘The mutations are in different complementation groups’. If the diploid has a mutant phenotype, then the mutations do not complement and are said to be in the same complementation group. This is considered strong evidence that the mutations are alleles. The definition of comple- mentation group is a set of noncomplementing mutations. The term complementa- tion group is synonymous with gene. Two conditions must bemet before one can carryout a complementation analysis on a series of mutants. First, the mutant strain can only contain a single mutation compared with the parental strain. Particularly when mutagenesis had been used to obtain the mutants, it is possible that more than oneDNA alteration was induced in an individual and these are involved in producing the mutant phenotype. Therefore, each mutant strain must be tested todemonstrate whether one or more genetic alterations are required to produce the mutant phenotype. To test this the mutant strain is crossed to a wild-type strain and tetrad analysis of the heterozygous diploid is carried out. If only a single alteration is required, then only tetrads with two mutant spores and two wild-type spores will be produced, as shown in Cross 1 in Chapter 1. But if two or more alterations are present, tetratype and nonparental ditype tetrads will be produced, as shown in Cross 3 in Chapter 1. What would be the phenotypes of the spores of a tetratype tetrad if the mutant strain containedtwo altered genes and both alterations were required to produce the mutant phenotype? What would be the phenotypes of the spores of a tetratype tetrad if the mutant straincontained two altered genes and eithermutation alone were sufficient to produce the mutant phenotype? Of course to carry out a cross between themutant and wild-type strainsthe strainsmust be of oppositemating type and should carry different nutritional mutationsto facilitatethe selection of diploids. But in other respects the two strains should ideally be isogenic except for any alterations required to produce the mutant phenotype. Usually, before undertaking a mutant hunt, the geneticist will construct an appropriate pair of isogenic (or congenic) haploid strains to be used as parental strains. The mutants isolated in one strain can then be mated to the parental strain of the opposite mating type to determine the number of mutant genes involved. As described above, the second requirement for a complementation analysis is that the mutations be loss of function alleles. In other words, only mutations that are recessive to the wild-type allele can be used. So, as a second step in the genetic analysis of mutants, mutant strains carrying a single mutant gene are crossed to a parental strain carrying the wild-type allele. If the mutant carries a recessive loss of functionmutation, then the heterozygous diploid (GENllgenl-34) will have the wild-type phenotype.This mutant allele canthen be used forcomplementation analysis. CO M PLEM ENTAT ION ANALYSIS COMPLEMENTATION 75 Cross 4 shows a complementation test for two mutant strains. Preliminary genetic analysis has shown that each strain contains only a single mutant gene and that the mutant allele is recessive.

Cross 4: Mutant strain 5 x Mutant strain 14 Diploidphenotype: Mutant

The result shown in Cross 4 indicates that the mutationin strain 5 and the mutation in strain 14 do not complement and thus are mutations in the same gene. If we call the gene GENI, then these mutations are alleles and one could now name them genl-5 and genl-14. This cross could be depicted as shown below.

Cross 4: genl-5 X genl-l4 (genotypes of parentalstrains) (mutant)(mutant) (phenotype of parentalstrains)

Diploid: genl-5 (genotype of diploid) genl -I 4 (mutant)(phenotype of diploid)

As a second test of whether or not the mutations are alleles, the researcher can determinethe segregation pattern of the allelesin the meiotic products of the diploid. If the two mutations are in the same gene, then recombination between the mutations will be relatively rare because they map so close to one another. Therefore, 100% of the time (or close to it) the two mutant genes will segregate to different spores producing a tetrad with four mutant spores (two mutant #5 spores and two mutant #14 spores). This situation is depicted in Cross 2 of Chapter 1). Cross 5 shows anothercomplementation test between mutantstrain 5, which carries the mutation genl-5, and another mutant strain. Mutant strain 4 contains only a single mutant gene and the mutant allele is recessive.

Cross 5: Mutant strain 5 x Mutant strain4 Diploidphenotype: Wild-type

The result shown in Cross 5 indicates that the mutation in strain 5 and the mutation in strain 4 complement and thus are mutations in different genes. We can then say that a different gene, GEN2, is mutant in strain 4. This cross could be depicted as shown below.

Cross 5: genl-5GEN2 X GENl gen2-4 (genotypes of parentalstrains) (mutant)(mutant)(phenotype of parentalstrains)

Diploid: --genl-5 gen2-4 (genotype of diploid) GENl GEN2 (wild-type) (phenotype of diploid) 76 GENETICTECHNIQUESBIOLOGICAL FOR RESEARCH If GENl and GEN2 are not linked, then the mutant genes will recombine pro- ducing recombinant meiotic products with the wild-type (GENI GEN2) and double mutant (genl-5 gen2-4) genotype. This is exhibited by the presence of tetratype and nonparentalditype tetrads when this diploid is subjected to tetrad analysis (see Cross 3 in Chapter 1). The frequency of each type of tetrad will depend on the frequency of recombination. If the two genes are completely unlinked, that is 50% recombination, the frequency of PD : TT : NPD tetrads will be 1 : 4 : 1. If there is any linkage, thenthe frequency of recombination isless than 50% and the relative number of PD tetrads will increase to greater than the expected 116 of the total number of tetrads analyzed. Ultimately, for crosses between two alleles, the number of PD tetrads will closely approach loo%, as is shown in Cross 1. One can calculate the map distance between two mutations (the frequency recombination multiplied by 100) using the following formula, which is correct for map distances up to 35 CM (Sherman & Wakem, 1991):

6 x # NPD tetrads + # TT tetrads map distance in CM = ~ 2 total # of tetrads

Thecombination of these twomethods, complementation analysis and tetrad analysis, should clearly indicate whether one is dealing with mutations in one or more genes. Either method alone is not as powerful, and therefore researchers do both tests. For example, if there are mutations in two very tightly linked genes, the mutations will complement, but recombination will be rare and most, if not all the tetrads will be PD. Such results would strongly suggest that one is dealing with mutations in two closely linked genes. In a complete complementation analysis, all the mutants are crossed to all of the other mutants. Often as a complementation group containing several mutant alleles is identified, one allele will be chosen as the representative of that complementation group and only this allele will be crossed to the other mutants. At the end of this process, all the mutants isolated in aparticular mutant selection/screen will be placed into complementation groups, i.e. genes. The researcher will have made a good start at determining the number of genes involved in the process of interest. If only a few genes have been identified with several mutant alleles, then the researcher will have some degree of confidence that the analysis saturated the genome and that new genes are not likely to be identified by the same selection/screening method. If many genes have been identified, several with only one mutant allele each, then it is likely that new genes will be identified if the same selection/screen is repeated. There are some special situations in which straightforwardinterpretation of a complementation test is misleading and the geneticist must be on the alert for such possibilities. Infrequently, mutant alleles of the same gene are able to complement, and produce a heterozygous diploidwith a wild-type like phenotype. This is referred toas intragenic complementation. Intrageniccomplementation can occur if the encoded polypeptide forms a multiple subunit protein composed of like subunits, such as a homodimer, or if it encodes a single polypeptide that carries out several distinctfunctions. In the case of thehomomultimeric protein, mutant subunits encoded by different mutant genes associate with one another in the multimeric protein and are able to accommodate each other’s mutant alteration in the mixed CO MPLEM ENT ATION ANALYSIS COMPLEMENTATION 77 multimer. When this happensthe mixed-mutant multimer complex has some functional activity, although it may not be completely normal. Another mechanism of intrageniccomplementation is possible if theprotein product of the gene has several distinct functions, such as two different enzyme activities. In this situation it is possible to obtain mutations that affect one of these activities while leaving the other function intact. In a heterozygous diploid, cells carrying two different mutations, each one affecting only one of the two functions, proteins capable of carrying out both enzyme activities will be produced, albeit in different molecules, and the cell should have the wild-type phenotype. Incontrast to intrageniccomplementation where mutations in the same gene complement, in a few instances mutations in different genes which are expected to complement do not. This phenomenon is referred to as nonallelic noncomplementa- tion. One explanation for this noncomplementation is that the two genes encode subunits of a heteromultimeric protein, and that the presence of a mutant alteration in either subunit destroys all function of the multimeric protein. Sort of, ‘one bad apple spoils the whole barrel’. Careful and thorough genetic analysis involving both complementation tests and genetic mapping of several mutant alleles is necessary to avoid the pitfalls of these potentially misleading situations.

REFERENCES

Benzer, S. (1995) Fine structure of a genetic region in bacteriophage. Proc. Natl Acad. Sci. USA 41: 344-354. Sherman, F. & P. Waken (1991) Mapping yeast genes. Methods Enzymol. 194: 38-57.