Complementation and allelism: the cis-trans test
Complementation and allelism, commonly investigated through the cis-trans test, are vital concepts in genetics that assess the relationship between different mutations within genes. Developed by Seymour Benzer in the 1950s using the T4 bacteriophage, the cis-trans test examines whether mutations in different strains of phage can compensate for each other when introduced together. The test determines if two mutations are in the same gene or different genes based on the resulting phenotype of the organism. If the mutations complement each other, the organism will display a wild-type phenotype; if not, it will exhibit a mutant phenotype.
This approach relies on the understanding that genes are structured sequences of nucleotides in DNA, and mutations can alter this sequence, potentially affecting protein production. Alleles can be in two configurations: cis (on the same chromosome) or trans (on opposite homologs), affecting their ability to produce normal phenotypes. The cis-trans test also facilitates the mapping of mutations and can reveal complex interactions known as pseudoalleles, where mutations occur in separate genes that interact functionally. Through these investigations, researchers have gained insights into gene structure, function, and the biochemical pathways that underpin various traits.
Complementation and allelism: the cis-trans test
Categories: Genetics; methods and techniques
The cis-trans test was developed by Seymour Benzer and his coworkers in the 1950’s. They worked with a bacteriophage (a virus that attacks only bacteria) called T4, studying one T4 viral gene called rII. Mutants of rII could be easily identified by the size of the plaques they made. A plaque is a clear circle in a continuous lawn of bacteria growing on nutrient agar which results when viruses attack and destroy the bacteria in that area.
Complementation (cis-trans) tests between mutations in different bacteriophage, or phage, strains which produce mutant plaques can be performed using a procedure called a spot test. Phages of one mutant strain are added to bacterial cells, then spread out on a plate containing nutrients and a solidifying agent (usually agar). Later, a drop of fluid containing phages with a different mutant strain is spotted onto the solid surface. In the area of the drop, some bacterial cells will be attacked by phages of both types. If the mutations complement each other, a normal plaque will result in that area. After looking at hundreds of different mutations, all of which had the same phenotype (that is, the expression of the gene) of large plaques, scientists at Benzer’s laboratory determined that the rII gene was actually made up of two functional groups (which they called cistrons) and that every mutant could be assigned to one or the other of these groups.
Genetic Basis
Genes are located on cellular structures called chromosomes. For many years, scientists thought that genes were lined up along the chromosomes like beads on a string, each bead representing a different gene, such as for seed shape in plants. If normal or wild-type alleles were represented by black beads, it was thought, a red or white or green bead would represent a changed, or mutant, allele. This analogy was popular for many years, and genes, like beads, were thought to be indivisible. The work of scientists in the 1940’s and 1950’s challenged this theory and led to its eventual demise. The cis-trans test was a key part of this work.
The basis of the cis-trans test is the functional role of the gene. It is now known that each gene is made up of a particular sequence of nucleotides in deoxyribonucleic acid (DNA), defining the nucleotide sequence of a strand of ribonucleic acid (RNA). This RNA may, in turn, be used to define the sequence of amino acids in a protein. A mutant allele is a stretch of DNA in which the nucleotide sequence has been altered, resulting in an altered RNA. The result may be an altered protein, or sometimes no protein at all.
In most higher organisms, chromosomes occur in pairs called homologs. Such pairing means every gene (except those on the sex chromosomes) is normally found in two copies. If one copy is mutated and the other gene makes a normal product that compensates, the mutation is said to be recessive. If both copies of the gene are mutated and no normal product is made, then the phenotype of the organism (that is, the expression of the gene) will be mutant.
Genetic Crosses
In the cis-trans test, genetic crosses are arranged to yield an organism in which two recessive mutant alleles to be tested are on opposite homologs. In other words, one chromosome of a pair will have one mutant allele, and the other chromosome will have the other mutant allele. The alleles are then said to be in a trans configuration. If the two alleles are mutations of the same gene, the phenotype of the organism will be a mutant one. If the alleles belong to separate genes, the organism will have a normal, or wild-type, phenotype. When alleles produce a normal phenotype, they are said to complement each other. If the mutant alleles are arranged in the cis configuration, so that both are on the same chromosome, the resulting phenotype will be normal, whether the alleles are in the same or different genes.
For example, harebells (Campanula) normally have blue flowers, the wild-type phenotype, but occasionally mutants have white flowers. If a cis-trans test is done with two white-flowered mutants, in some cases the resulting progeny have blue flowers. This shows that the mutations in these two mutants are in different genes and thus display complementation. It is known that the blue pigment is produced by a biochemical pathway with two steps involving two enzymes that modify a colorless molecule. In other cases when a cis-trans test is done, all the progeny are white-flowered. This occurs when both mutants have mutations in the same gene, knocking out the activity of one of the enzymes, and thus complementation does not occur.
Recombination
Before the structure of DNA was understood, the gene was also defined as the basic unit of recombination; recombination within a gene was not thought to occur. In the analogy of genes as beads on a string, recombination was thought to involve the breakage and reunion of the string at positions between beads, never within them. It is now known that, in the process of recombination, pieces of chromosomes break off and rejoin in new combinations, although the rules that this process follows are not fully understood.
Recombination is essential to the cis-trans test. Only through recombination can cis configurations be obtained. The frequency of the occurrence of recombination is related to the closeness of two points on a chromosome. If recombination is desired between two regions that are very close, the chance that crossing over will occur is very low. Recombination can even be used to map mutations within a gene (a process called fine structure analysis), but it is a very long process, requiring the analysis of an extremely large number of progeny because the distance separating mutations within a gene will be very small.
Using the cis-trans test and recombination together led to the discovery of a small group of mutations called pseudoalleles. When two pseudoalleles are examined using the cis-trans test, an organism in trans configuration will have a mutant phenotype, leading to the conclusion that the alleles belong to the same gene. When recombination is used to try to map these mutations, however, it turns out that they are in separate structural genes. Pseudoalleles, then, are found in separate structural genes that interact in some functions.
Several examples of interaction between pseudoalleles can be found in Drosophila (fruit flies). In one such example, the two genes postbithorax (pbx) and bithorax (bx) are both involved in determining the normal formation of the fly’s balancing organs (halteres), a pair of winglike structures that help the fly in its flight. These genes are adjacent on the chromosome but are structurally distinct. The fact that the gene products must interact is revealed by the results of the cis-trans test; when mutants for these genes are placed in trans configuration, the phenotype of the fly is mutant.
Bibliography
Benzer, Seymour. “The Fine Structure of the Gene.” Scientific American 206 (January, 1962): 70-84. The classic review article on the investigation of the rII gene of the T4 bacteriophage, written by the scientist who headed the investigations. Designed for readers with some familiarity with viruses and bacteria.
Hawkins, John D. Gene Structure and Expression. 3d ed. New York: Cambridge University Press, 1996. A concise guide to gene structure and function, giving an overview of recent advances in the field from both theoretical and practical points of view.
Russell, P. J. Genetics. 5th ed. Menlo Park, Calif.: Benjamin Cummings, 1998. A well-written, intermediate-level college text with many touches of humor. Complementation analysis is discussed in the chapter on genetic fine structure and gene function. Includes index, bibliography, and glossary.
Suzuki, D., A. Griffiths, J. Miller, and R. Lewontin. An Introduction to Genetic Analysis. 7th ed. New York: W. H. Freeman, 2000. This intermediate-level college text reviews the important concepts in genetics and presents the experimental work that led to the development of important theories. Chapter 12, “The Nature of the Gene,” gives an in-depth analysis of Seymour Benzer’s viral work. Chapter 15, “The Manipulation of DNA,” also explains many of the important techniques of molecular genetics.
Weaver, R. F., and P. W. Hedrick. Genetics. 3d ed. New York: McGraw-Hill, 1996. Written for college audiences, the discussion is nevertheless readable by advanced high school students. Unlike many texts, it includes full-color photographs and diagrams. Chapter 13 discusses the cis-trans test using bacterial genes as an example. Seymour Benzer’s work with rII is covered briefly in the same chapter. Recombination and potential mechanisms of how it works on the molecular level are clearly explained in chapter 7.