Complementation testing
Complementation testing is a genetic method used to ascertain whether two mutations occur within the same gene. This technique is particularly valuable in situations where multiple mutations produce similar phenotypes, making it challenging to determine if they affect the same or different genes. By crossing organisms carrying different mutations, researchers can observe the phenotypes of the offspring. If the offspring display the mutant phenotype, the mutations are likely in the same gene and do not complement each other. Conversely, if the offspring exhibit a normal phenotype, the mutations complement each other, indicating they are in different genes.
Complementation testing has important applications in studying genetic diseases, particularly inborn errors of metabolism. Historically, researchers like Sir Archibald Garrod and George Beadle utilized complementation to explore how specific mutations affect metabolic pathways. This foundational work led to the formulation of the "one gene-one enzyme hypothesis," which has evolved into the notion that each gene encodes a single polypeptide. Moreover, complementation testing can aid in identifying alleles of a gene, thereby enhancing the understanding of its function and significance in various biological processes.
Complementation testing
SIGNIFICANCE: Complementation testing is used to determine whether or not two mutations occur within the same gene.
Finding Mutations
Most traits are the result of products from several genes. Mutations at any one of these genes may produce the same mutant phenotype. If the same mutant phenotype is observed in two different strains of an organism, there is no way, using simple observation, to determine whether this shared mutant phenotype represents a mutation in the same or different genes, or loci. One way of solving this problem is through complementation testing. If the mutations are alleles of the same locus, then a cross between mutant individuals from the two strains will only produce offspring with the mutant phenotype. In genetic terms, they fail to complement each other and are therefore members of the same complementation group. If from the same cross, all the offspring are normal; the two mutations are at the same locus and they are said to complement each other. Researchers often want to define multiple alleles of a single gene in order to understand the gene’s function better.
Often a researcher is interested in the genetic control of a particular biological process, such as the biochemistry of eye color in fruit flies. As a first step, researchers often screen large numbers of individuals to find abnormal phenotypes involving the process in which they are interested. For instance, researchers studying eye color in fruit flies may screen hundreds of thousands of fruit flies for abnormal eye colors. Complementation testing is then used to organize the mutations into complementation groups.
Complementation Testing and Inborn Errors of Metabolism
Human genetic diseases that affect the function of cellular enzymes are known as inborn errors of metabolism and were defined by Sir Archibald Garrod long before DNA was determined to be the hereditary material. Garrod studied families with alkaptonuria, a disease that causes urine to turn dark upon exposure to air. He determined that this biochemical defect was inherited in a simple Mendelian fashion.
George Beadle and Edward Tatum studied mutant strains of Neurospora and expanded on Garrod’s work. They used radiation to generate random mutations that resulted in strains of Neurospora that could not grow without specific nutritional supplements (essentially creating yeast with inborn errors of metabolism). Some of the mutant strains required the addition of a specific amino acid to the media. Each mutant strain had its own specific requirements for growth, and each strain was shown to have a single defective step in a metabolic pathway. When strains that had different defects were grown together, they were able to correct each other’s metabolic defect. This correction was termed metabolic complementation. Using complementation tests, Beadle and Tatum were able to establish the number of genes required for a particular pathway. These studies formed the basis for the “one gene-one enzyme hypothesis”: Each gene encodes a single enzyme required for a single step in a metabolic pathway. This hypothesis has since been renamed the “one gene-one polypeptide hypothesis” because some enzymes consist of multiple polypeptides, each of which is encoded by a single gene.
The Biochemical Basis for Complementation Testing
Complementation testing is useful for locating and identifying the genes affected by recessive or loss-of-function alleles. A researcher crosses two organisms that are each homozygous for a recessive mutation. If these two alleles affect the same gene, they will not complement each other, because the first-generation (F1) offspring will inherit one mutant copy of the gene from one parent and a second mutant copy of the gene from the other parent, thus having no normal copies of the gene. If the mutations are alleles of two different genes, genes A and B, the F1 offspring will receive a normal copy of A and a mutant copy of B from one parent and a mutant copy of A and a normal copy of B from the other, thus having one normal copy of each of the two genes and having a wild-type (normal) phenotype. The mutations are said to complement each other.
If a scientist is interested in a particular gene, obtaining as many alleles of that gene as possible will lead to a better understanding of how the gene works and what parts of the gene are essential for function. One way to identify new alleles of a gene is through an F1 noncomplementation screen. In this type of screen, the researcher treats the model organism with radiation or chemicals to increase the rate of mutation. Any individuals from the screen that segregate the desired phenotype (white eyes, for example) in a Mendelian fashion are crossed with individuals carrying a known mutation in the gene of interest. If the progeny of this cross have white eyes (the mutant phenotype), then the two mutations have failed to complement each other and are most likely alleles of the same gene. Such noncomplementation screens have been used to identify genes involved in a wide variety of processes ranging from embryo development in fruit flies to spermatogeneis in Caenorhabditis elegans. In a 2024 study, complementation testing was also used to trace specific genes that caused a fear response in human behavior.
Key terms
- allelea form of a gene; each gene (locus) in most organisms occurs as two copies called alleles
- cistrona unit of DNA that is equivalent to a gene; it encodes a single polypeptide
- inborn errors of metabolismconditions that result from defective activity of an enzyme or enzymes involved in the synthesis, conversion, or breakdown of important molecules within cells
- locuspl. loci ): the location of a gene, often used as a more precise way to refer to a gene; each locus occurs as two copies called alleles
Bibliography
Chen, Patrick B., et al. “Complementation Testing Identifies Genes Mediating Effects at Quantitative Trait Loci Underlying Fear-Related Behavior." Cell Genomics, vol. 4, no. 5, 8 May 2024, www.cell.com/cell-genomics/fulltext/S2666-979X(24)00101-0. Accessed 6 Sept. 2024.
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