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Modern Genetic Analysis Chapter 6. Gene Interaction
A Diagnostic Test for Alleles

Many genetic research programs are undertaken to attempt to understand the genes that contribute to one particular biological process. Such an analysis begins with a collection of related mutant phenotypes centered on that particular process. For example, if a geneticist was interested in the genes determining locomotion in a nematode worm, the genetic dissection would begin by isolating a set of different mutants with defective locomotion. An important task is to determine how many different genes are represented by the alleles that determine the related phenotypes, because this defines the set of genes that affect the process under study. Hence it is necessary to have a test to find out if the mutations are alleles of one gene or of different genes.

The allelism test that finds widest application is the complementation test, which is illustrated in the following example. Consider a species of harebell in which the wild-type flower color is blue. Let's assume that by application of mutagenic radiation we have induced three white-petaled mutants and that they are all homozygous pure-breeding strains. We can call the mutant strains $, £, and ¥, using currency symbols to avoid prejudicing our thinking concerning dominance. When crossed to wild type, each mutant gives the same results in the F1 and F2, as follows:

In each case the results show that the mutant condition is determined by the recessive allele of a single gene. However, are they three alleles of one gene, or of two or three genes? The question can be answered by asking if the mutants complement each other. First, let us define complementation.

MESSAGE
Complementation is the production of a wild-type phenotype when two recessive mutant alleles are united in the same cell.

(The demonstration of the recessive nature of individual mutants is a crucial result that allows us to proceed with a complementation test. Dominant mutations cannot be used in a complementation test.)

In a diploid organism the complementation test is performed by intercrossing homozygous recessive mutants two at a time and observing whether or not the progeny have wild-type phenotype. If recessive mutations represent alleles of the same gene, then obviously they will not complement because they both represent lost gene function. Such alleles can be thought of generally as a and a", using primes to distinguish between two different mutant alleles of a gene whose wild-type allele is a+. These alleles could have different mutant sites but would be functionally identical. The heterozygote a/a" would be

However, two recessive mutations in different genes would have wild-type function provided by the respective wild-type alleles. Here we can name the genes a1 and a2, after their mutant alleles. Heterozygotes would be a1/+ ; +/a2 (unlinked genes) or a1 +/+ a2 (linked genes), and we can diagram them as follows:

Let us return to the harebell example and intercross the mutants to test for complementation. Assume the results of intercrossing mutants $, £, and ¥ are as follows:

From this set of results we would conclude that mutants $ and £ must be caused by alleles of one gene (say w1) because they do not complement; but ¥ must be caused by a mutant allele of another gene (w2).

The molecular explanation of such results is often in terms of biochemical pathways in the cell. How does complementation work at the molecular level? Although it is conventional to say that it is mutants that complement, in fact the active agents in complementation are the proteins produced by the wild-type alleles. The normal blue color of the flower is caused by a blue pigment called anthocyanin. Pigments are chemicals that absorb certain parts of the visible spectrum; in the case of the harebell the anthocyanin absorbs all wavelengths except blue, which is reflected into the eye of the observer. However, this anthocyanin is made from chemical precursors that are not pigments; that is, they do not absorb light of any specific wavelength and simply reflect back the white light of the sun to the observer, giving a white appearance. The blue pigment is the end product of a series of biochemical conversions of nonpigments. Each step is catalyzed by a specific enzyme coded by a specific gene. We can accommodate the results with a pathway as follows:

A mutation in either of the genes in homozygous condition will lead to the accumulation of a precursor, which will simply make the plant white. Now, the mutant designations can be written as follows:

However, in practice the subscript symbols would be dropped and the genotypes written

Hence an F1 from $ × £ will be

which will have two defective alleles for w1 and therefore be blocked at step 1. Even though enzyme 2 is fully functional, it has no substrate to act on, so no blue pigment will be produced and the phenotype will be white.

However the F1s from the other crosses will have the wild-type alleles for both the enzymes needed to take the interconversions to the final blue product. Their genotypes will be

Hence we see why complementation is actually a result of the cooperative interaction of the wild-type alleles of the two genes. Figure 6-1 shows a summary diagram of the interaction of the complementing and noncomplementing white mutants.

In a haploid organism the complementation test cannot be performed by intercrossing. In fungi an alternative way to test complementation is to make a heterokaryon (Figure 6-2). Fungal cells fuse readily, and when two different strains fuse, the nuclei from the different strains come to occupy one cell, which is called a heterokaryon (Greek: "different kernels"). The nuclei in a heterokaryon generally do not fuse. In one sense this merely mimics a diploid condition. Assume that in different strains we have mutations in two different genes conferring the same mutant phenotype, for example arginine requirement. We can call these genes arg-1 and arg-2. The two strains, whose genotypes can be represented as arg-1 . arg-2+ and arg-1+ . arg-2, can be fused to form a heterokaryon with the two nuclei in a common cytoplasm:

Because gene expression takes place in a common cytoplasm, the two wild-type alleles can exert their dominant effect and cooperate to produce a heterokaryon of wild-type phenotype. In other words, the two mutations complement just as they would in a diploid. If the mutations had been alleles of the same gene, no complementation would have occurred.

MESSAGE
When two independently derived recessive mutant alleles producing similar recessive phenotypes fail to complement, the alleles must be of the same gene. If they do complement, they are from different genes.

© 1999 by W. H. Freeman and Company. All rights reserved.