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McClintock and the Ac/Ds Transposable Elements of Corn Barbara McClintock was the first scientist to predict that transposable elements, mobile pieces of the genetic material (DNA), were present in eukaryotic genomes. She performed her work on corn and specifically followed seed color phenotypes. Before we discuss her experiments, it is necessary to describe the morphology of the corn seed and the parental source of the genes which control its phenotype. Higher plants are characterized by a double fertilization of the female gametophyte. After gametogenesis, the female gametophyte is located in the ovule and contains eight nuclei. Those which are important for this discussion are the egg cell nuclei which is near the location where the pollen tube enters the ovule and two polar nuclei that are in the center of the ovule. The pollen grain (male gametophyte) is found in the anther and contains three nuclei, of which the two sperm nuclei are of importance for fertilization. After the pollen tube enters the female gametophyte, one of the sperm nuclei unites with the egg cell nuclei to form the 2n zygote that will develop into the embryo. The other sperm nuclei unites with the two polar nuclei to form the 3n endosperm tissue. For corn, the outer layer of cells of the endosperm is the aleurone layer. McClintock discovered transposable elements by analyzing genetic stocks of corn that were phenotypically unstable. In particular, she was analyzing genes that control the color of the aleurone layer of the endosperm. Remember that this tissue is triploid (3n). The genes that she was following were located on the short arm of chromosome 9 of corn and were involved in the development of the color of the seed. The genetic map of this region and the allelic designations follow. _____________________________________________ _____________________________________________O C Bz Ds Genes Studied by McClintock CI = dominant allele that prevents color from being expressed in the aleurone layer C = recessive allele that leads to color development in the aleurone layer Bz = dominant allele that produces purple aleurone color bz = recessive allele that produces a dark brown to purple-brown aleurone color Ds = a genetic location where chromosome breakage occurs Homozygous stocks were created and CC bzbz -- females (without ds, denoted by the dash) were mated with CICI BzBz DsDs males. The aleurone layer of the endosperm would thus have the genotype CICC Bzbzbz --Ds. Because of the presence of the inhibitor allele, the aleurone layer was expected to be colorless. For many of the kernels this was the case, but a few kernels had dark brown colored sectors on an otherwise colorless background. How could this have occurred? McClintock concluded that in some manner the CI and Bz alleles were lost because chromosome breakage had occurred at the Ds locus. But why the sectoring? This breakage apparently did not occur during gamete formation, but had occurred after fertilization and during the development of the seed. This breakage and loss of genes occurred in a single cell, but all cells that developed from mitotic division of that cell did not contain the inhibitor gene, so the color expression was controlled by the recessive bz allele in those cells. Female gametes: _____________________________________________O C bz Male gametes: _____________________________________________O CI Bz Ds The following is the expected chromosomal composition of triploid endosperm. Because of the dominant CI allele the endosperm should be colorless without breakage. _____________________________________________O (from the female) C bz _____________________________________________O (from the female) C bz _____________________________________________O (from the male) CI Bz Ds But if breakage at ds occurred, then the genotype of the endosperm would be: _____________________________________________O (from the female) C bz _____________________________________________O (from the female) C bz //_____________O (from the male) CI Bz and any cells with this genotype would be dark brown in color. Breakage at Ds had been established by McClintock prior to performing these experiments. The designation Ds was short for dissociation or a locus were breakage of chromosomes occurred. But after crossing with a number of different genetic stocks, she realized that Ds alone could not induce the breakage. A second factor, Ac, short for activator, was also necessary. (Thus, some genetic stocks contained Ac whereas other stocks did not contain that locus.) This system is called a two-element system and historically has been called the Ac/Ds system. Additional genetic stocks were analyzed by McClintock, and she determined that in the presence of Ac, Ds could move locations as well as cause breakages. She was able to isolate a corn line where Ds had moved into the normal Bz allele and caused a mutation in that gene. But as was mentioned this only occurred when Ac was present. Furthermore, when this new line was used and Ac was present, the Ds element was shown to move out of the Bz locus and reversion to the original phenotype was detected. This mutated allele was designated bzm1. But in the absence of Ac, bzm1 was a stable allele. Another unstable Bz allele was found that contained an Ac insertion and was designated bzm2. One difference between this allele and bzm1 was its higher rate of transposition and reversion back to the original phenotype. So what conclusions can be drawn from these experiments and observations: Ds requires some factor provided by Ac to move, whereas Ac is independent Because of their relationship, Ac is termed an autonomous element and Ds a non- autonomous element. Because both Ac and Ds can move, they are called transposable genetic elements. Somewhat astonishingly, McClintock was able to draw all of these conclusions by genetic analysis of appropriate corn stocks and her in depth understanding of the cytogenetics of corn. Her primary core of research was performed between 1944 and 1950 and was met with a great deal of skepticism. Today, though transposable elements are recognized as important components of many genomes and may have played important roles in evolution. Once the tools of molecular biology became a part of research in plant genetics, one of the goals was to clone both Ac and Ds. As expected these two transposable elements appear to be related. In general, all Ac elements are identical, 4563 base pairs (bp) in length. Ds elements are Ac elements that have undergone deletions. The factor that stimulates the movement of Ac is a transposase protein encoded by the element. Deletions of Ac that created Ds eliminated all or part of this transposase. This lack of transposase activity accounts for the inability of Ds elements to move in the absence of Ac. The transposase that is encoded by Ac elements can move throughout the cell and excise any Ds or Ac element. Because of this ability, the Ac/Ds transposase is said to be trans-acting. What molecular features are required for an element to move? First, the transposase must be present. This protein works by recognizing sequences that are in common to both Ac and Ds elements. All the elements contain a short inverted repeat sequence (11 bp) at each end that appear to be essential for transposition of the element. Another common feature that results from insertion is an 8-bp direct repeat that is generated on either end of the element. These sequences remain after excision and are footprints that mark were the element has been. Sometimes after reversion, the expression of the allele is changed because the direct repeats altered the properties of the final protein product. Thus, the presence of the transposable element in the allele changes the phenotype produced by the allele. Transposition of the element out of the allele, can also generate a new allele with new activities. In summary, the molecular features of the maize Ac/Ds system are: Ac is 4563 bp in length contain 11-bp inverted repeats at the ends 8-bp direct repeats of target DNA are generated Ds are truncated versions of Ac Copyright © 1997. Phillip McClean