The First Mutagenesis Assay
Before DNA sequencing became widespread, most mutations could only be detected by their effects on the phenotype of the organism. Many mutations are recessive, however, and do not affect the phenotype if present in only one allele. Hermann Muller, who pioneered the study of mutations, overcame this problem by focusing on the X chromosome in his studies of the genetics of Drosophila, the fruit fly. While females have two X chromosomes, males have only one, so any mutated gene carried on the X chromosome is expressed in males, even if it is recessive. Hence recessive lethal mutations on the X chromosome kill any male inheriting them, but would not kill a female. Muller's method examined all of the genes on the X chromosome that could mutate to give a recessive lethal mutation. Muller used X rays to generate mutants. X rays are a very high-energy form of radiation, and break the DNA at numerous points. The method is shown in Figure 1.
Muller treated adult males with X rays and mated them to females who carried one copy of a specially prepared X chromosome, called ClB. This chromosome had a gene to prevent crossing over (C), which kept the chromosome intact; a lethal recessive gene (l) to kill any males that inherit it; and a dominant "bar eye" gene (B) that resulted in a distinctive phenotypic change, making it easy to find female flies that inherited it.
Muller mated X-ray treated males with ClB-carrying females. All female offspring from this crossbreeding received one treated X chromosome from the male (which might or might not have carried a lethal recessive gene). They also received one X from the female, either normal or ClB. He selected only the bar-eyed females for further mating. To determine which of these females carried an X-ray induced lethal recessive, he separated each female into a separate jar, and examined their offspring.
Three types of males were created in this cross, depending on what type of X chromosome they inherited. Males inheriting the ClB chromosome died, due to the presence of the l gene. Males inheriting an X-ray-treated X chromosome with a lethal recessive died. Males inheriting an X-ray-treated X chromosome without a lethal recessive lived. Therefore, any jar with live males indicated that the mother did not carry a lethal recessive. Any jars with no males indicates the mother carried a lethal recessive, originally induced in the X-ray-treated male. The analysis was rapid because an experienced person could examine a bottle of flies and see at a glance if there were males present. Subsequent studies showed that this method tested almost 1,000 genes simultaneously, thus making it practical to use when detecting rare mutations. Unfortunately the breeding takes quite a lot of time, so this assay has now fallen largely into disuse, despite its historical importance.