Discovery of Transposable Elements as Controlling Elements
The discovery of transposable elements by Barbara McClintock is a remarkable story of careful study and insightful analysis in genetics. Long before the chemical structure of genes was known, she observed that genetic determinants, called controlling elements, in maize were moving from one location to another. The controlling elements regulate the expression of other genes. The families of controlling elements are now recognized as members of the class of transposable elements that move through DNA intermediates. However, McClintock’s proposal that the controlling elements were mobile was not widely accepted for a very long time. Despite her extensive observations published in the 1930’s through the 1950’s, the interpretation that genetic elements could move was perhaps too novel. Indeed, the notion that transposable elements are active in a wide range of species as not widely accepted until the 1980’s, and new evidence continues to mount that transposable elements are more common than previously thought.
McClintock’s seminal observations relied on two complementary approaches to understanding chromosome structure and function. One was cytological, using microscopy to examine the structure of chromosomes in corn, and the other used genetics to follow the fates of the chromosomes. A full exploration of the discovery of transposable elements is the subject of excellent books. In this section, we will examine a few examples of the type of studies that were done, to give some impression of the care and insight of the work.
In essence, McClintock showed that certain crosses between maize cultivars (or strains) resulted in large numbers of mutable loci, i.e. the frequency of change at those loci is much higher than observed in other crosses. Her studies of the cultivars with mutable loci revealed a genetic element termed “Dissociation”, or Ds. Chromosome breaks occurred at the Ds locus; these could be seen cytologically, using a microscope to examine chromosome spreads from individual germ cells (sporocytes). The frequency and timing of these breaks is controlled by another locus, called “Activator” or Ac. In following crosses of the progeny, the position of Ds-mediated breaks changed, arguing that the Dselement had moved, or transposed. That was the basic argument for transposition.
Frequent chromosome breaks at Ds
The studies of Dson chromosome 9 illustrate the combination of morphological examination of chromosome structure plus genetic analysis to show that the controlling elements were mobile in the genome. Chromosome 9 of maize has a knob at the end of its short arm, making it easy to identify when chromosome spreads are examined in the microscope. In some versions of chromosome 9, a long stretch of densely staining heterochromatin extends beyond the knob, forming a hook (but shown as a green oval in Fig. 9.2). These morphologically distinct versions of the same chromosome can have different sets of alleles for the genes on this chromosome. As diagrammed in Fig. 9.2, several genes affecting the appearance of corn kernels are on this chromosome. The colorlessgene has three alleles we will consider: the recessive c allele confers no color, the Callele (dominant to c) makes the kernel colored, and the I allele, which is dominant to C, confers no color. The recessive allele shmakes the kernel look shrunken, whereas the dominant Shis nonshrunken. The recessive bzconfers a bronze phenotype, whereas the dominant Bzdoes not. The recessive wxgives the kernel a waxy appearance, whereas the dominant Wx makes the kernel starchy. Of course, all the phenotypes stated for recessive alleles are for the homozygous or hemizygous (only one allele present, e.g. because the other is deleted) states. Thus different versions of chromosome 9 that have a distinctive appearance in the microscope (knob or extended heterochromatin at the ends) confer different phenotypes on progeny.
Figure 9.2.Two homologs of chromosome 9 can be distinguished both by appearance and genetic determinants. The short of chromosome 9 can have either a knob or extended heterochromatin, denoted by the green circle and the elongated oval, respectively. The alleles of each of the genes diagrammed confer different phenotypes. The yellow circle is the centromere (CEN). Ds is the dissociation element that leads to chromosome breaks.
The two homologs will pair to form a bivalent during the pachytene phase of meiosis I. Ordinarily, the two homologs will form a continuous complex with no disruptions, as shown in panel 3 and 3a of Fig. 9.3. However, when Dsis on the short arm of chromosome 9 and an Acelement is also present in the genome, a break in one of the chromosomes in the pair can be seen when spreads of chromosomes are examined in the microscope (panels 4, 4a, 5 and 5a). One can identify chromosome 9 specifically because of the knob or extended heterochromatin at its end. In panels 4 and 4a, a break has occurred in the knob chromosome (with the dominant alleles diagrammed in Fig. 9.2), leaving the other homolog intact, with the recessive alleles and marked by the extended heterochromatin). Both a break and a crossover occurred in the chromosome pair shown in panels 5 and 5a. In a given strain, the break usually occurred in the same position, so the genetic element at the site of the break was called “dissociation”, or Ds.
Figure 9.3. Cytological examination in the microscope reveals breaks on morphologically marked chromosomes. The figure shows photomicrographs (panels 3, 4 and 5) and interpretative drawings (panels 3a, 4a and 5a) of paired homologous chromosomes at the pachytene phase of meiosis. The telomere at the end of the short arm of chromosome 9 (labeled a in the pictures) can be either a darkly staining spot, called a knob, or an elongated hook. The centromere is labeled b and the breaks are labeled c. These images are adapted from a 1952 paper from McClintock in the Cold Spring Harbor Symposium on Quantitative Biology.
These effects of these frequent breaks in the chromosomes could be seen phenotypically when the sporocytes (e.g. pollen grains) with Ds and Ac were used to fertilize ova of a known genotype. For instance, pollen from a plant homozygous for the “top” chromosome in Fig. 9.4.A. will carry the dominant alleles (indicated by capitalized names) for all the loci shown. When this pollen is used to fertilize an ovum that has the recessive alleles along chromosome 9, the resulting corn kernel will show the phenotypes specified by the dominant alleles. However, if the chromosome with the dominant alleles also has a Dselement, and Acis present in the genome, the chromosome will break in some of the cells making up the kernel as some stage in development. Then the region between Dsand the telomere will be lost from this chromosome, and the phenotype of the progeny cells will be determined by the recessive alleles on the other chromosome. For example, the phenotype of the kernel outlined in Fig. 9.4.A. will be colorless, nonshrunken and nonwaxy (starchy), but the sector of the kernel derived from a cell in which a break occurred at Ds will be colored, shrunken and waxy. In more detail, I is dominant to C (which itself is dominant to c; hence the capital letter). This gives a colorless seed when the chromosome is intact, but after the break, I is lost and Cis left, generating a colored phenotype. Similarly, prior to the break the starch will not be waxy (Wx is dominant), but after the break one sees waxy starch because only the recessive wxallele is present.
Figure 9.4. Breaks at Ds can reveal previously hidden phenotypes of recessive alleles (in the presence of Ac). A. Prior to the break, the dominant alleles along the chromosome with Ds(IShBzWx, shown at the top) determine the phenotype. [The part of the corn kernel showing the phenotypes studied is actually triploid, resulting from fertilizing a diploid ovum with a haploid pollen grain. For this discussion the diploid ovum is homozygous recessive, and only one copy is shown, C sh bz wx.] After the chromosome breaks at Ds, which occurs frequently in the presence f Ac, the phenotype will be determined by the recessive alleles thus revealed, C sh bz wx. B. Kernels with variegating color. The chromosome breaks in some but not all cells, and only those with the broken cells show the new phenotypes. All the progeny of the cells with a broken chromosome are located adjacent to each other, resulting in a patch of cells with the same new phenotype. Thus the new phenotype is variegating across the kernel. The kernel shown in panel 10 is colorless, determined by the Iallele. Panels 11-13 show patches of colored kernel, representing patches of cells in which the I allele has been deleted because of the chromosome break and revealing the effect of the Callele. B was adapted from McClintock in the Cold Spring Harbor Symposium on Quantitative Biology.
These frequent breaks occurring at different times in different cells derived from the fertilized ovum can produce a sectored, patched or stippled appearance to the corn kernel, as illustrated in Fig. 9.4.B. The phenotype differs in the various parts of the kernel, even though all the cells are derived from the same parental cell (i.e. the kernel is clonal). This differing phenotype in a clonal tissue is called variegation. Each sector is the product of the expansion of one cell. When a chromosome breaks in that cell, thereby removing the effect of a dominant allele I that was making the seed colorless, then all the progeny in that sector would be colored (from the effects of the C allele in the example in Fig. 9.4.B.).
Variegating phenotypes can be caused by breaks such as those described here, but in other cases they result from modifications to the regulation of genes. Variegation is a fairly common occurrence, and is especially visible in flower petals, as illustrated for the wildflowers in Fig. 9.5.
Figure 9.5. Variegation in sectors of wildflower petals. A wildflower blooms all over the middle Atlantic states in the USA in late May and June. My neighbors call this wild flox. It is an invasive plant, but it is pretty when it blooms. It has two predominant flower colors, purple (A) and white (B). However, a casual examination of the plants reveals sectored petals at a moderate frequency (C-E). This is a variegating phenotype of unknown origin. It can produce white sectors on purple petals (C) or purple sectors on white petals (D, E).
Question 9.1.How does this phenotype in Fig. 9.5, panels C-E, differ from partial dominance, e.g. with the purple allele dominant and the white allele recessive?
Ds can appear at new locations
By following several generations of a maize cultivar with Ds on chromosome 9, McClintock observed that Ds could move to new locations. As outlined in Fig. 9.6, chromosomal rearrangements associated with Dsactivity can appear at several different positions on chromosome 9. If, e.g., Ds were centromeric to Wxin one generation, but it was between Iand Shin a subsequent generation, the simplest explanation is that it had moved. These observations are the basis for the notion that Ds is transposable.
Figure 9.6. Ds activity can appear at new locations on chromosome 9.
How does one know that Ds is present at different locations on chromosome 9? The effects of breaking the chromosome (Fig. 9.2 and 9.4) depend on where Ds is. The position of the observable break (e.g. bottom panel of Fig. 9.3) and the genetic consequences in terms of which recessive allele are revealed, will differ depending on where Ds is.
Question 9.2.What phenotype in kernels would result if the second chromosome after the arrow in Fig. 9.6 were in a heteroduplex with the recessive chromosome shown in Fig. 9.4.A, and Ac were also present?
The example of a single Ds affecting all the genes telomeric to it on chromosome 9 shows a particular controlling element can simultaneously regulate the expression of genes involved in a variety of biochemical pathways. The Ac element is needed to activate the mobility of any Ds element, regardless of its chromosomal location. Thus controlling elements can operate independently of the chromosomal location of the controlling element. These observations show that the controlling element is distinct from the genes whose expression is being regulated.
The movement of Ds to new locations on chromosome 9 is associated with other types of recombinations that involve breaks, including duplications and inversions. Other types of transposable elements also cause inversions and duplications in their vicinity when they move.
Figure 9.7. The appearance of Ds at a new location is associated with duplications and inversions. Some of the rearranged chromosomes found in progeny in which Ds had moved are shown.