Mutagenesis

To overcome the problem of the rarity of mutations, researchers induce mutations with a variety of agents. Hermann Muller was the first to do this when, in 1927, he used X rays on fruit flies (Drosophila) to increase the mutation rate by more than 100-fold. Other high-energy forms of radiation can also be used to create mutations.

The first chemical to be recognized as a mutagen was mustard gas, which had been developed during World War I, but not tested until World War II by Auerbach and Robson, at the University of Edinburgh. Since then a wide variety of chemicals have been discovered that are also mutagenic. Some induce mutations at any point in the cell cycle, by disrupting DNA structure. Others only act during DNA replication. Called base analogs, these latter chemicals have structures similar to the bases found in DNA, and are incorporated instead of the normal base.

Transposable genetic elements (also called transposons, or "jumping genes") can also induce mutations. These elements insert randomly into the genome, and may disrupt gene function if inserted into a gene or its promoter. Finding the organism with a disrupted gene is made easier if the transposon carries with it a reporter gene whose product can be identified, or a selectable marker that allows the transformed cells to live while non-transformed ones die. (The use of reporter genes and selectable markers are techniques used in genetic analysis in the laboratory.) The transposon sequence itself serves as a molecular tag. Thus, if the target gene (the gene being studied) is interrupted, finding the transposon allows the researcher to find the gene.

All of the above methods disrupt genes randomly. However, specific genes can also be targeted, for "site-directed mutagenesis," if their sequence is known. Using the known sequence, a matching DNA sequence is inserted into a single-stranded vector. Short, complementary, partial sequences containing the desired mutation are then synthesized. These are allowed to pair up, and DNA polymerase is then used to complete the complementary strand. Further replication amplifies he number of copies of the mutant. In bacteria, the mutant gene can be placed on a plasmid for transformation of the bacteria. The bacteria make the mutant protein, and the effect of the mutation can then be studied. This is a key tool in studying how amino acid sequences affect protein structure, since individual amino acids can be changed, one at a time.

In eukaryotes, the mutant gene can be inserted into the chromosome of an experimental organism by "homologous recombination," a system in which the mutant gene switches places with the normal chromosomal gene. Such techniques can "knock out" and "knock in" genes bearing the desired mutations.