Complementation Test Genetics Example Essay

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 were 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 mutations that determine the related phenotypes, because this number 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 having the widest application is the complementation test, which is illustrated in the following example.

Consider a species of harebell (Campanula) in which the wild-type flower color is blue. Let’s assume that, by applying mutagenic radiation, we have induced three white-petalled mutants and that they are available as homozygous pure-breeding strains. We can call the mutant strains $, £, and ¥, using currency symbols so that we do not prejudice our thinking concerning dominance. When crossed with 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. ed as follows:

MESSAGE

Complementation is the production of a wild-type phenotype when two haploid genomes bearing different recessive mutations are united in the same cell.

Figure

A harebell plant (Campanula species). (Gregory G. Dimijian/ Photo Researchers.)

(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. The next step is to observe whether the progeny have the wild-type phenotype.

This unites the two mutations as haploid gametes to form a diploid nucleus in one cell (the zygote). If recessive mutations represent alleles of the same gene, then they will not complement, because both mutations represent lost gene function. Such alleles can be thought of generally as a′ and a′, by 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 they would be functionally identical (that is, both nonfunctional). The heterozygotea′/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. We can represent the heterozygotes as follows, depending on whether the genes are on the same or different chromosomes:

Let us return to the harebell example and intercross the mutants to unite the mutant alleles to test for complementation. We can assume that the results of intercrossing mutants $, £, and ¥ are as follows:

From this set of results, we can 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 relation to biochemical pathways in the cell. How does complementation work at the molecular level? Although the convention is to say that it is the 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 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 encoded by a specific gene. We can accommodate the results with a pathway as follows:

Amutation in either of the genes in homozygous condition will lead to the accumulation of a precursor that will simply make the plant white. Now the mutant designations could be written as follows:

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

Hence an F1 from $ × £ will be:

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

The F1s from the other crosses, however, 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 the reason why complementation is actually a result of the cooperative interaction of the wild-type alleles of the two genes. Figure 4-1 is a summary diagram of the interaction of the complementing and noncomplementing white mutants.

Figure 4-1

The molecular basis of genetic complementation. Three phenotypically identical white mutants—$, £, and ¥—are intercrossed to form heterozygotes whose phenotypes reveal whether the mutations complement each other. (Only (more...)

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 4-2). Fungal cells fuse readily and, when two different strains fuse, the haploid nuclei from the different strains occupy one cell, which is called a heterokaryon (Greek; different kernels). The nuclei in a heterokaryon generally do not fuse. In one sense, this condition is a “mimic” diploid. Assume that, in different strains, there are mutations in two different genes conferring the same mutantphenotype—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:

Figure 4-2

Formation of a heterokaryon of Neurospora, demonstrating both complementation and recessiveness. Vegetative cells of this normally haploid fungus can fuse, allowing the nuclei from the two strains to intermingle within the same cytoplasm. If each strain (more...)

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 wildtype 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, there would have been no complementation.

MESSAGE

When two independently derived recessive mutant alleles producing similar recessive phenotypes fail to complement, the alleles must be of the same gene.

Complementation test, also called cis-trans test, in genetics, test for determining whether two mutations associated with a specific phenotype represent two different forms of the same gene (alleles) or are variations of two different genes. The complementation test is relevant for recessive traits (traits normally not present in the phenotype due to masking by a dominant allele). In instances when two parent organisms each carry two mutant genes in a homozygous recessive state, causing the recessive trait to be expressed, the complementation test can determine whether the recessive trait will be expressed in the next generation.

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When two mutations occur in different genes, they are said to be complementary, because the heterozygote condition rescues the function otherwise lost in the homozygous recessive state. Hence, the term complementation test is used to describe the process to test for gene function in recessive allelism. The alternative name cis-trans test describes the two central components of the test. The terms cis and trans refer to the relationship of the two mutations, with cis used to describe mutations occurring on the same chromosome and trans used to describe mutations occurring on different chromosomes. The cis portion of the complementation test essentially acts as a control and involves creating heterozygotes (one mutated chromosome and one wild-type, or normal, chromosome) such that one parent bears both mutations. In the cis test, a functional protein is always produced regardless of whether both mutations are on the same gene or on different genes. The trans test involves creating heterozygotes with different mutations from different parents. In this case a functional protein is produced only if the mutations are on different genes.

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