Although transposable elements were first discovered in maize, the first eukaryotic elements to be characterized at the molecular level were isolated from mutant yeast and Drosophila genes. Eukaryotic transposable elements fall into two classes: class 1 retrotransposons and class 2 DNA transposons. The first class to be isolated, the retrotransposons, are not at all like the prokaryotic IS elements and transposable elements.

The laboratory of Gerry Fink was among the first to use yeast as a model organism to study eukaryotic gene regulation. Through the years, he and his colleagues isolated thousands of mutations in the HIS4 gene, which encodes one of the enzymes in the pathway leading to the synthesis of the amino acid histidine.

They isolated more than 1500 spontaneous HIS4 mutants and found that two of them had an unstable mutant phenotype. The unstable mutants (called pseudorevertants) were more than 1000 times as likely to revert to a phenotype that was similar to wild type as the other HIS4 mutants. Symbolically, we say that these unstable mutants reverted from His⁻ to His+ (wild types have a superscript plus sign, whereas mutants have a superscript minus sign). Like the E. coli gal^– mutants, these yeast mutants were found to harbor a large DNA insertion in the HIS4 gene. The insertion turned out to be very similar to one of a group of transposable elements already characterized in yeast, called the Ty elements. There are, in fact, about 35 copies of the inserted element, called Ty1, in the yeast genome.

Cloning of the elements from these mutant alleles led to the surprising discovery that the insertions did not look at all like bacterial IS elements or transposons. Instead, they resembled a well-characterized class of animal viruses called retroviruses. A retrovirus is a single-stranded RNA virus that employs a double-stranded DNA intermediate for replication. The RNA is copied into DNA by the enzyme reverse transcriptase. The double-stranded DNA is integrated into host chromosomes, from which it is transcribed to produce the RNA viral genome and proteins that form new viral particles. When integrated into host chromosomes as double-stranded DNA, the double-stranded DNA copy of the retroviral genome is called a provirus. The life cycle of a typical retrovirus is shown in Figure 15-11. Some retroviruses, such as mouse mammary tumor virus (MMTV) and Rous sarcoma virus (RSV), are responsible for the induction of cancerous tumors. For MMTV, this happens when it inserts randomly into the genome next to a gene whose altered expression leads to cancer.

Figure 15-12 shows the similarity in structure and gene content of a retrovirus and the Ty1 element isolated from the HIS4 mutants. Both are flanked by long terminal repeat (LTR) sequences that are several hundred base pairs long. Retroviruses encode at least three proteins that take part in viral replication: the products of the gag, pol, and env genes. The gag-encoded protein has a role in the maturation of the RNA genome, pol encodes the all-important reverse transcriptase, and env encodes the structural protein that surrounds the virus. This protein is necessary for the virus to leave the cell to infect other cells. Interestingly, Ty1 elements have genes related to gag and pol but not env. These features led to the hypothesis that, like retroviruses, Ty1 elements are transcribed into RNA transcripts that are copied into double-stranded DNA by the reverse transcriptase. However, unlike retroviruses, Ty1 elements cannot leave the cell because they do not encode env. Instead, the double-stranded DNA copies are inserted back into the genome of the same cell. These steps are diagrammed in Figure 15-13.

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In 1985, David Garfinkel, Jef Boeke, and Gerald Fink showed that, like retroviruses, Ty elements do in fact transpose through an RNA intermediate. Figure 15-14 diagrams their experimental design. They began by altering a yeast Ty1 element, cloned on a plasmid. First, near one end of an element, they inserted a promoter that can be activated by the addition of galactose to the medium. Second, they introduced an intron from another yeast gene into the coding region of the Ty transposon.

The addition of galactose greatly increases the frequency of transposition of the altered Ty element. This increased frequency suggests the participation of RNA because galactose stimulates the transcription of Ty DNA into RNA, beginning at the galactose-sensitive promoter. The key experimental result, however, is the fate of the transposed Ty DNA. The researchers found that the intron had been removed from the transposed Ty DNA. Because introns are spliced only in the course of RNA processing (see Chapter 8), the transposed Ty DNA must have been copied from an RNA intermediate. The conclusion was that RNA is transcribed from the original Ty element and spliced. The spliced mRNA undergoes reverse transcription back into double-stranded DNA, which is then integrated into the yeast chromosome. Transposable elements that employ reverse transcriptase to transpose through an RNA intermediate are termed retrotransposons. They are also known as class 1 transposable elements. Retrotransposons such as Ty1 that have long terminal repeats at their ends are called LTR-retrotransposons, and the mechanism they use to transpose is called "copy and paste" to distinguish them from the cut-and-paste mechanism that characterizes most DNA transposable elements.

Several spontaneous mutations isolated through the years in Drosophila also were shown to contain retrotransposon insertions. The copia-like elements of Drosophila are structurally similar to Ty1 elements and appear at 10 to 100 positions in the Drosophila genome (see Figure 15-12c). Certain classic Drosophila mutations result from the insertion of copia-like and other elements. For example, the white-apricot (w ^a) mutation for eye color is caused by the insertion of an element of the copia family into the white locus. The insertion of LTR-retrotransposons into plant genes (including maize) also has been shown to contribute to spontaneous mutations in this kingdom.

Before we leave retrotransposons (we will return to them later in this chapter), there is one question that needs to be answered. Recall that the first LTR-retrotransposon was discovered in an unstable His⁻ strain of yeast that reverted frequently to His^*. However, we have just seen that LTR-retrotransposons, unlike most DNA transposable elements, do not excise when they transpose. What, then, is responsible for this allele’s ~1000-fold increase in reversion frequency when compared to other His^– alleles? The answer is shown in Figure 15-15, which shows that the Ty1 element in the His^– allele is located in the promoter region of the His gene, where it prevents gene transcription. In contrast, the revertants contain a single copy of the LTR, called a solo LTR. This much smaller insertion does not interfere with the transcription of the His gene. The solo LTR is the product of recombination between the identical LTRs, which results in the deletion of the rest of the element (see Chapters 4 and 16 for more on recombination). Solo LTRs are a very common feature in the genomes of virtually all eukaryotes, indicating the importance of this process. The sequenced yeast genome contains more than fivefold as many solo LTRs as complete Ty1 elements.

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Some mobile elements found in eukaryotes appear to transpose by mechanisms similar to those in bacteria. As illustrated in Figure 15-8 for IS elements and transposons, the entity that inserts into a new position in the genome is either the element itself or a copy of the element. Elements that transpose in this manner are called class 2 elements, or DNA transposons. The first transposable elements discovered by McClintock in maize are now known to be DNA transposons. However, the first DNA transposons to be molecularly characterized were the P elements in Drosophila.

P elements Of all the transposable elements in Drosophila, the most intriguing and useful to geneticists are the P elements. The full-size P element resembles the simple transposons of bacteria in that its ends are short (31-bp) inverted repeats and it encodes a single protein—the transposase that is responsible for its mobilization (Figure 15-16). The P elements vary in size, ranging from 0.5 to 2.9 kb in length. This size difference is due to the presence of many defective P elements from which parts of the middle of the element—encoding the transposase gene—have been deleted.

P elements were discovered by Margaret Kidwell, who was studying hybrid dysgenesis—a phenomenon that occurs when females from laboratory strains of D. melanogaster are mated with males derived from natural populations. In such crosses, the laboratory stocks are said to possess an M cytotype (cell type), and the natural stocks are said to possess a P cytotype. In a cross of M (female) × P (male), the progeny show a range of surprising phenotypes that are manifested in the germ line, including sterility, a high mutation rate, and a high frequency of chromosomal aberration and nondisjunction (Figure 15-17). These hybrid progeny are dysgenic, or biologically deficient (hence, the expression hybrid dysgenesis). Interestingly, the reciprocal cross, P (female) × M (male), produces no dysgenic offspring. An important observation is that a large percentage of the dysgenically induced mutations are unstable; that is, they revert to wild type or to other mutant alleles at very high frequencies. This instability is generally restricted to the germ line of an individual fly possessing an M cytotype by a mechanism explained below.

The unstable Drosophila mutants had similarities to the unstable maize mutants characterized by McClintock. Investigators hypothesized that the dysgenic mutations are caused by the insertion of transposable elements into specific genes, thereby rendering them inactive. According to this view, reversion would usually result from the excision of these inserted sequences. This hypothesis has been critically tested by isolating unstable dysgenic mutations at the eye-color locus white. Most of the mutations were found to be caused by the insertion of a transposable element into the white⁺ gene. The element, called the P element, was found to be present in from 30 to 50 copies per genome in P strains but to be completely absent in M strains.

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Why do P elements not cause trouble in P strains? The simple answer is that P-element transposition is repressed in P strains. At first it was thought that repression was due to a protein repressor that was in P but not M strains. This model is no longer favored. Instead, geneticists now think that all the transposase genes in P elements are silenced in P strains. The genes are activated in the F₁ generation as shown in Figure 15-18. Gene silencing has been discussed previously (see Chapters 8 and 12) and will be revisited at the end of this chapter. For some reason, most laboratory strains have no P elements, and consequently the silencing mechanism is not activated. In hybrids from the cross M (female, no P elements) × P (male, P elements), the P elements in the newly formed zygote are in a silencing-free environment. The P elements derived from the male genome can now transpose throughout the diploid genome, causing a variety of damage as they insert into genes and cause mutations. These molecular events are expressed as the various manifestations of hybrid dysgenesis. On the other hand, P (female) × M (male) crosses do not result in dysgenesis because, presumably, the egg cytoplasm contains the components required for silencing the P-element transposase.

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An intriguing question remains unanswered: Why do laboratory strains lack P elements, whereas strains in the wild have P elements? One hypothesis is that most of the current laboratory strains descended from the original isolates taken from the wild by Morgan and his students almost a century ago. At some point between the capture of those original strains and the present, P elements spread through natural populations but not through laboratory strains. This difference was not noticed until wild strains were again captured and mated with laboratory strains.

Although we don’t know exactly how P elements have spread, it is clear that transposable elements can spread rapidly from a few individual members of a population. In this regard, the spread of P elements resembles the spread of transposons carrying resistance genes to formerly susceptible bacterial populations.

Maize transposable elements revisited Although the causative agent responsible for unstable mutants was first shown genetically to be transposable elements in maize, it was almost 50 years before the maize Ac and Ds elements were isolated and shown to be related to DNA transposons in bacteria and in other eukaryotes. Like the P element of Drosophila, Ac has terminal inverted repeats and encodes a single protein, the transposase. The nonautonomous Ds element does not encode transposase and thus cannot transpose on its own. When Ac is in the genome, its transposase can bind to both ends of Ac or Ds elements and promote their transposition (Figure 15-19).

As noted earlier in the chapter, Ac and Ds are members of a single transposon family, and there are other families of transposable elements in maize. Each family contains an autonomous element encoding a transposase that can move elements in the same family but cannot move elements in other families because the transposase can bind only to the ends of family members.

Although some organisms such as yeast have no DNA transposons, elements structurally similar to the P and Ac elements have been isolated from many plant and animal species.

Quite apart from their interest as a genetic phenomenon, DNA transposons have become major tools used by geneticists working with a variety of organisms. Their mobility has been exploited to tag genes for cloning and to insert transgenes. The P element in Drosophila provides one of the best examples of how geneticists exploit the properties of transposable elements in eukaryotes.

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Using P elements to tag genes for cloning P elements can be used to create mutations by insertion, to mark the position of genes, and to facilitate the cloning of genes. P elements inserted into genes in vivo disrupt genes at random, creating mutants with different phenotypes. Fruit flies with interesting mutant phenotypes can be selected for cloning of the mutant gene, which is marked by the presence of the P element, a method termed transposon tagging. After the interrupted gene has been cloned, fragments from the mutant allele can be used as a probe to isolate the wild-type gene.

Using P elements to insert genes Gerald Rubin and Allan Spradling showed that P-element DNA can be an effective vehicle for transferring donor genes into the germ line of a recipient fly. They devised the following experimental procedure (Figure 15-20). Suppose the goal is to transfer the allele ry⁺, which confers a characteristic eye color, into the fly genome. The recipient genotype is homozygous for the rosy (ry⁻) mutation. From this strain, embryos are collected at the completion of about nine nuclear divisions. At this stage, the embryo is one multinucleate cell, and the nuclei destined to form the germ cells are clustered at one end. (P elements mobilize only in germ-line cells.) Two types of DNA are injected into embryos of this type. The first is a bacterial plasmid carrying a defective P element into which the ry⁺ gene has been inserted. The defective P element resembles the maize Ds element in that it does not encode transposase but still has the ends that bind transposase and allow transposition. This deleted element is not able to transpose, and so, as mentioned earlier, a helper plasmid encoding transposase but without the terminal repeats (so it cannot transpose) also is injected. Flies developing from these embryos are phenotypically still rosy mutants, but their offspring include a large proportion of ry⁺ flies. In situ hybridization confirmed that the ry⁺ gene, together with the deleted P element, was inserted into one of several distinct chromosome locations. None appeared exactly at the normal locus of the rosy gene. These new ry⁺ genes are found to be inherited in a stable, Mendelian fashion.

Because the P element can transpose only in Drosophila, these applications are restricted to these flies. In contrast, the maize Ac element is able to transpose after its introduction into the genomes of many plant species, including the mustard weed Arabidopsis, lettuce, carrot, rice, and barley. Like P elements, Ac has been engineered by geneticists for use in gene isolation by transposon tagging. In this way, Ac, the first transposable element discovered by Barbara McClintock, serves as an important tool of plant geneticists more than 50 years later.

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