Complementation (genetics)

Complementation refers to the capacity of a segment of genetic material (eg DNA) to rescue the phenotype of a mutation.  It shows that a copy of the gene affected by the mutation is contained within the segment of genetic material and provides an important criterion for deciding which mutations affect which genes.

Simple complementation test Complementation can be assessed by  mating or crossing  strains of an organism that each carry mutations.  When the mutations in question are homozygous and recessive,  complementation will ordinarily result in a normal (or "wild-type") phenotype if the mutations are in different genes (intergenic complementation). When the mutations are in different genes, each strain's genome supplies the wild-type allele to "complement" the mutated allele of the other strain's genome. Since the mutations are recessive, the offspring that is heterozygous mutant at each gene will display the wild-type phenotype. When the mutations affect the same gene, neither genome can supply a wild type allele and a mutant phenotype results.  Such a simple complementation may sometimes produce offspring with a weaker phenotype than one or both parental strains.  This is still indicates that both mutations likely affect the same gene, since it indicates that the offspring lack completely wild type gene function.  Simple complementation tests (ie mating or crossing strains homozygous for recessive mutations) provide a convenient practical approach that works in most cases to assign mutations to the same or different genes without the molecular information.

The term complementation is also applied to similar phenomena achieved without mating or crossing. For example, a mutation can be complemented by a gene encoded on a piece of DNA that is introduced into the cell using molecular biology techniques,  rather than introduced through mating.

Exceptions to the complementation rule can occur.  These include intragenic complementation, and non-allelic non-complementation (see also sections further below). In intragenic complementation, mutations affecting the same gene can nevertheless complement, which sometimes happens when each mutation affects a discrete function of a multi-functional gene product. Non-allelic non-complementation  occurs when mutations affecting different genes can fail to complement, eg when they affect genes whose products interact or have inter-dependent functions.  Non-allelic non-complementation can be distinguished using a cis-trans test.  A Cis-trans test is also useful for determining allelism of dominant mutations, which is not always revealed by simple complementation tests.

Cis-trans test If the phenotype of organisms bearing two mutations linked on the same chromosome (ie in cis) is the same as the phenotype of organisms bearing the two mutations on opposite  homologous chromosome pair (ie in trans), then the two mutations are likely to affect different genes.  This is because when the mutations are in different genes, both the cis and trans genotypes have one mutant and one wild type copy of each gene and the same phenotype is expected. When the mutations are in cis in the same gene, the diploid organism has one wild type and one doubly-mutated copy of that gene, but when the mutations in the same gene are in trans, the organism has no wild type copies of the gene, and the phenotype may not be the same.  Cis-trans tests can reveal allelism between dominant and recessive mutations, and they can distinguish allelism from non-allelic non-complementation.

Cis-trans test can also yield exceptions.  For example, the cis and trans combinations of the Drosophila mutations Cbx¹ and pbx¹ yield the same phenotype, although both are alleles of the Ultrabithorax gene.  The molecular basis for this anomalous behavior was explained by Bender et al.^[1]    

The American geneticist EB Lewis may have been the first person to compare the phenotypes of mutations in cis and in trans, using the fruitfly Drosophila,^[2] but the modern understanding of complementation as a means of assigning mutations to genes is very much due to Benzer’s work with bacteriophage,^[3]

For a simple example of a complementation test, suppose a geneticist is interested in studying two strains of white-eyed flies of the species Drosophila melanogaster , more commonly known as the common fruit fly. In this species, wild-type flies have red eyes, and eye color is known to be related to two genes, A and B. Each of these genes has two alleles, a dominant one that codes for a working protein (A and B respectively) and a recessive one that codes for a malfunctioning protein (a and b respectively). Since both proteins are necessary for the synthesis of red pigmentation in the eyes, if a given fly is homozygous for either a or b, it will have white eyes.

Knowing this, the geneticist may perform a complementation test on two separately obtained strains of pure-breeding white-eyed flies. The test is performed by crossing two flies, one from each strain. If the resulting progeny have red eyes, the two strains are said to complement; if the progeny have white eyes, they do not.

If the strains complement, we imagine that one strain must have a genotype aa BB and the other AA bb, which yields the genotype AaBb when crossed. In other words, each strain is homozygous for a different deficiency that produces the same phenotype. If the strains do not complement, they both must have genotypes 'aaBB', 'AAbb', or 'aabb'. In other words, they are both homozygous for the same deficiency, which obviously will produce the same phenotype.

Complementation tests can also be carried out with haploid eukaryotes such as fungi, with bacteria, and with viruses such as bacteriophage.^[4] Research on the fungus Neurospora crassa led to the development of the one-gene-one-enzyme concept that provided the foundation for the subsequent development of molecular genetics.^{[5] [6]} The complementation test was one of the main tools used in the early Neurospora work, because it was easy to do, and allowed the investigator to determine whether any two nutritional mutants were defective in the same or different genes.

The complementation test was also used in the early development of molecular genetics when bacteriophage T4 was one of the main objects of study.^[7] In this case the test depends on mixed infections of host bacterial cells with two different bacteriophage mutant types. Its use was key to defining most of the genes of the virus, and provided the foundation for the study of such fundamental processes as DNA replication and repair, and how molecular machines are constructed.

Heterosis is the tendency for hybrid individuals to exceed their purebred parents in size and vigor. The phenomenon has long been known in animals and plants. Heterosis appears to be largely due to genetic complementation, that is the masking of deleterious recessive alleles in hybrid individuals.

In general, the two fundamental aspects of sexual reproduction in eukaryotes are meiosis and outcrossing. These two aspects have been proposed to have two natural selective advantages, respectively. Meiosis is proposed to be adaptive because it facilitates recombinational repair of DNA damages that are otherwise difficult to repair. Outcrossing is proposed to be adaptive because it facilitates complementation, that is the masking of deleterious recessive alleles ^[8] (also see heterosis). The benefit of masking deleterious alleles has been proposed to be a major factor in the maintenance of sexual reproduction among eukaryotes. Further, the selective advantage of complementation that arises from outcrossing may largely account for the general avoidance of inbreeding in nature (e.g. see articles kin recognition, inbreeding depression, and incest taboo).^[9]

Used by quantitative genetics to uncover recessive mutants. Here one takes deficiencies and crosses them to a haplotype that is believed to contain the recessive mutant.

These rules (patterns) are not without exceptions. Non-allelic mutants may occasionally fail to complement (this is known as "non-allelic non-complementation" or "unlinked non-complementation"). This is an uncommon occurrence that depends on the type of mutants being investigated. Two mutations, for example, could be synthetically dominant negative. Transvection is another instance, in which a heterozygous combination of two alleles with mutations in distinct sections of the gene complement one other to restore a wild-type phenotype.^[10]

When complementation between two mutants defective in the same gene is measured, it is generally found that there is either no complementation or the complementation phenotype is intermediate between the mutant and wild-type phenotypes. Intragenic complementation (also called inter-allelic complementation) has been demonstrated in many different genes in a variety of organisms including the fungi Neurospora crassa , Saccharomyces cerevisiae, and Schizosaccharomyces pombe ; the bacterium Salmonella typhimurium; and the virus bacteriophage T4.^[11] In several such studies, numerous mutations defective in the same gene were isolated and mapped in a linear order based on recombination frequencies to form a genetic map of the gene. Separately, the mutants were tested in pairwise combinations to measure complementation. An analysis of the results from such studies led to the conclusion that intragenic complementation, in general, arises from the interaction of differently defective polypeptide monomers to form an aggregate called a "multimer."^[12] Genes that encode multimer-forming polypeptides appear to be common. One interpretation of the data is that polypeptide monomers are often aligned in the multimer in such a way that mutant polypeptides defective at nearby sites in the genetic map tend to form a mixed multimer that functions poorly, whereas mutant polypeptides defective at distant sites tend to form a mixed multimer that functions more effectively. The intermolecular forces likely responsible for self-recognition and multimer formation were discussed by Jehle.^[13]

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