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Modern Genetic Analysis Chapter 13. Transposable Genetic Elements
Controlling Elements in Maize

In 1938, Marcus Rhoades analyzed an ear of Mexican black corn. The ear came from a selfing of a pure-breeding pigmented genotype, but it showed a surprising modified Mendelian dihybrid segregation ratio of 12:3:1 among pigmented, dotted, and colorless kernels. Analysis showed that two events had occurred at unlinked loci. At one locus, a pigment gene A1 had mutated to a1, an allele for the colorless phenotype; at another locus, a dominant allele Dt (Dotted) had appeared. The effect of Dt was to produce pigmented dots in the otherwise colorless phenotype of a1 /a1 (Figure 13-22). Thus, the original line was very probably A1 /A1 ; dt /dt, and the mutations generated an A1 /a1 ; Dt /dt plant, which on selfing gave the observed ration of progeny.

But what was causing the dotted phenotype? A reverse mutation of a1 A1 in somatic cells would be an obvious possibility, but the large numbers of dots in the Dotted kernels would require extremely high reversion rates. Using special stocks, Rhoades was able to find anthers in the flowers of a1 /a1 ; Dt / plants that showed patches of pigment (Figure 13-23 on the following page). He reasoned that these anthers might contain pollen grains bearing the reverted pigment genotype, and he used the pollen from these anthers to fertilize a1 /a1 tested females. Sure enough, some of the progeny were completely pigmented, showing that each dot in the parental plants was in fact the phenotypic manifestation of a genetic reversion event. Thus, a1 is one of the first known examples of an unstable mutant allelean allele for which reverse mutation occurs at a very high rate. However, the allelic instability is dependent on the presence of the unlinked Dt gene. Once the reverse mutations occur, they are stable; the Dt gene can be crossed out of the line with no loss of the A1 character. This would not be surprising if the a1 phenotype arose from the insertion of a defective transposable element that by itself is unable to move. In the presence of a transfactor produced by the Dt locus, however, the element could move, yielding reversion to A1. The A1 allele would remain stable in the absence of Dt.

McClintock's Experiments: The Ds Element

In the 1950s, Barbara McClintock demonstrated an analogous situation in another study of corn. She found a genetic factor Ds (Dissociation) that causes a high tendency toward chromosome breakage at the location at which it appears. These breaks can be located either cytologically (Figure 13-24a) or genetically by the uncovering of recessive genes (Figure 13-24b). This action of Ds is another kind of instability. Once again, this instability proves to be dependent on the presence of an unlinked gene, Ac (Activator), in the same way that the instability of a1 is dependent on Dt.

McClintock found it impossible to map Ac. In some plants, it mapped to one position; in other plants of the same line, it mapped to different positions. As if this were not enough of a curiosity, the Ds locus itself (Figure 13-24) was constantly changing position on the chromosome arm, as indicated by the differing phenotypes of the variegated sections of the seeds (as different recessive gene combinations were uncovered in a system such as the one illustrated in Figure 13-24b).

The wanderings of the Ds element take on new meaning for us in the context of this chapter when we consider the results of the following cross:

Here C allows color expression and Ds+ and Ac+ indicate the lack of the element. Most of the kernels from this cross were of the expected types (Figure 13-25), but one exceptional kernel was very interesting. In Figure 13-25, the first seed shows the normal solid pigment pattern owing to the presence of the dominant C allele. The second seed shows the same basic background pigmentation, but with the expected white mottling caused by the loss of the C allele through chromosome breakage in some of the cell lines within the seed, with the resultant expression of the recessive c. Because of the clonal nature of cell growth in the seed, the size of a white patch is an indication of when in the seed's development the breakage occurred. A small white area suggests that the break came late in development, because it gave rise to only a small number of affected cells. A large patch suggests an early break, because many descendant cells are affected. The bottom seed in Figure 13-25 indicates expression of the c allele at the very beginning of development, inasmuch as the background is white, not pigmented. However, the presence of pigmented blotches on a white background suggests a reversible process at work that allows the C allele to be reexpressed. Chromosomal breakage could not be the explanation in this case, because, upon breakage, the C alleles are left on acentric fragments that are lost in mitosis, and, therefore, the white phenotype is not reversible in such cases. The white coloration in the bottom seed appears to be the result of a second type of action in which the Ds gene transposes into the C allele, disrupting its function but not inducing the chromosome to break. If the Ds allele is then excised and transposes elsewhere, the C allele regains its function, yielding the pigmented cells that form the patches. The Ac allele is still required, but in this case it has the effect of mediating a reversible instability at the C locus. If Ac is crossed out of the line, the cu allele becomes a stable mutant.

The analogy of this system with the a1 Dt system is obvious. Perhaps the earlier situation also is due to the insertion of a Ds-like element into the A1 gene. It is natural to ask whether a1 will respond to Ac or cu will respond to Dt. The answer is no; some kind of specificity prevents this cross-activation of mutational instability.

The wx (waxy) Locus

The Ds element can wander not only into the middle of the C gene, but also into other genes, rendering them unstable mutants dependent on Ac. One such locus, wx (waxy), has been the subject of an intense study on the effects of the Ds element. Oliver Nelson paired many unstable waxy alleles in the absence of the Ac mutation. In such wxm1 / wxm2 heterozygotes, he looked for rare wild-type Wx recombinants by staining the pollen with KI-I2 reagent, which stains Wx pollen black and wx pollen red. By counting the frequency of Wx pollen grains in each kind of heterozygote, Nelson was able to do fine-structure recombination mapping of the waxy gene. He showed that the different "mutable waxy" mutant alleles are in fact due to the insertion of the Ds element in different positions in the gene. Continuing the experiment, he allowed the Wx-bearing pollen to fertilize wx plants and produce rare Wx kernels, which could be detected by staining sliced-off slivers. The Wx kernels were then raised into plants, and the exchange of flanking markers expressed in the adults showed that the Wx pollen grains had arisen from chromosome exchange.

General Characteristics of Controlling Elements

Several systems like a1-Dt and Ds-Ac have now been found in corn. Each shows similar action, having a target gene that is inactivated, presumably by the insertion of some receptor element into it, and a distant regulator gene that maintains the mutational instability of the locus, presumably through its ability to "unhook" the receptor element from the target locus and return the locus to normal function. The receptor and the regulator are termed controlling elements.

In the examples considered so far, the unstable allele is said to be nonautonomous: it can revert only in the presence of the regulator. Sometimes, however, a system such as the Ac-Ds system can produce an unstable allele that is autonomous. Such mutants are recognizable because they show Mendelian ratios (such as 3:1 for pigmented to dotted) that apparently are independent of any other element. In fact, such alleles appear to be caused by the insertion of Ac itself into the target gene. An allele of this type can subsequently be transformed into a nonautonomous allele. In such cases, the nonautonomy seems to result from the spontaneous generation of a Ds element from the inserted Ac element. In other words, Ds is in all likelihood an incomplete version of Ac itself.

Figure 13-26 summarizes the overall behavior of the controlling elements in maize as inferred from genetic data. Note that the mutation events that gave rise to Rhoades's original ratio are nicely explained by this model: a nonautonomous Ds-like element is generated from an Ac-like progenitor; transposition of the Ds-like element into the A1 gene produces an inactive but mutationally unstable allele a1. Molecular studies in the past few years on Ac, Ds, and other controlling elements in maize have confirmed McClintock's genetic model.

MESSAGE
Controlling elements in maize can inactivate a gene in which they reside, cause chromosome breaks, and transpose to new locations within the genome. Complete elements can perform these functions unaided; other forms with partial deletions can transpose only with the help of a complete element elsewhere in the genome. Figures 13-27 and 13-28 show examples of the effects of transposons in maize and similar effects in the snapdragon.

© 1999 by W. H. Freeman and Company. All rights reserved.