User:Jelly Bean MD/Complementation

In genetics, complementation corresponds to the production of a wild-type phenotype by uniting two haploid genomes bearing different recessive mutations in the same cell. When the progeny are not wild-type, the recessive mutations must be alleles of the same gene. In other words, since both alleles of one gene bear the recessive mutations, a nonfunctional enzyme in the biosynthetic pathway is encoded. As a consequence, the organism is incapable of catalyzing the conversion of one precursor molecule into another in order to ultimately reach the substance responsible for the phenotype.

In the case of the fungus Neurospora crassa, complementation testing involves the fusion of two haploid organism to form one heterokaryon, bringing the mutant alleles together. The nuclei of the two different strains (e.g., auxotrophic arg-1 and arg-2 mutants) do not generally fuse; however, because the gene products are made in common cytoplasm, the two wild-type alleles can exert their dominant effect and cooperate to produce a heterokaryon of wild-type phenotype.

Neurospora and the arginine biosynthetic pathway

In order to fully understand the above example, one must be aware of the biosynthetic pathway of arginine, an amino acid that is essential for the survival of Neurospora. When the latter fungi undergo highly ionizing irradiation, mutations appear in their DNA. The mutagenized haploid Neurospora are crossed with wild-type organisms of the opposite mating type to form fruiting bodies. Some of the resulting octads contain auxotrophic mutants. However, in order to identify them, the microscopic ascospores are dissected and transferred one by one to culture tubes. First, a "pool" of organisms is created by keeping them on complete medium. A partial quantity is then transferred to minimal medium for identification of mutants. Cultures that fail to grow on minimal medium are subsequently tested on a variety of supplemented media. In the case of Neurospora auxotrophic mutants, it was noted that the addition of arginine restored growth.

It was then hypothesized that the ability to make arginine involved a biosynthetic pathway, and three distinct types of auxotrophic mutants were further identified: arg-1, arg-2, and arg-3. The production of arginine begins with the enzymatic conversion of precursor molecules to ornithine, which is then converted into the biochemically related molecule citrulline. This latter compound undergoes an enzymatic reaction to yield arginine. In other words, the production of arginine from the very beginning with the precursor molecules requires a total of three enzymes, which are encoded by genes. In arg-1 mutants, the first enzyme, which catalyzes the conversion of the precursor molecules to ornithine, is defective. Consequently, in order to produce arginine, the medium must be supplemented with ornithine or citrulline. The arg-2 mutants, on the other hand, have defects in the enzyme responsible for the second biochemical reaction in the biosynthetic pathway (i.e., the conversion of ornithine to citrulline). As such, the mutants much be supplemented with either citrulline or arginine in order to survive. The third class of mutants, arg-3, have a faulty enzyme responsible for the third reaction, that is, the conversion of citrulline to arginine. In this case, the mutants must imperatively be supplemented with arginine for survival.

Complementation testing can become useful when dealing with auxotrophic mutants. A simple example is the crossing of arg-2 with arg-3 mutants, which are both defective for different enzymes in the arginine biosynthetic pathway. Such a cross results in the obtention of a heterokaryon that grows without arginine.