An Introduction to Genetic Analysis
Chapter 20
Transposable Genetic Elements
Chapter 20
Transposable Genetic Elements
Key Concepts
A series of genetic elements can occasionally move, or transpose, from one position on a
chromosome to another position on the same chromosome or on a different chromosome.
In bacteria, insertion sequences, transposons, and phage mu are examples of transposable
genetic elements.
Transposable elements can mediate chromosomal rearrangements.
In higher cells, transposable elements have been extensively characterized in yeast,
Drosophila, and maize and in mammalian systems.
In eukaryotes, some transposable elements utilize an RNA intermediate during transposition,
whereas, in prokaryotes, transposition is exclusively at the DNA level.
Introduction
Starting in the 1930s, genetic studies of maize, undertaken independently by Barbara
McClintock and Marcus Rhoades, yielded results that greatly upset the classical genetic
picture of genes residing only at fixed loci on the main chromosome. The research literature
began to carry reports suggesting the existence of genetic elements of the main chromosomes
that can somehow mobilize themselves and move from one location to another. These
findings were viewed with skepticism for many years, but it is now clear that such mobile
elements are widespread in nature.
A variety of colorful names (some of which help to describe their respective properties) have
been applied to these genetic elements: controlling elements, cassettes, jumping genes, roving
genes, mobile genes, mobile genetic elements, and transposons. We choose the term
transposable genetic element, which is formally most correct and embraces the entire family
of types. The term transposition has long been used in genetics to describe transfer of
chromosomal segments from one position to another in major structural rearrangements. In
the present context, what is being transposed seems to be a gene or a small number of linked
genes or a gene-sized fragment. Any genetic entity of this size can be called a genetic
element.
Transposable genetic elements can move to new positions within the same chromosome or
even to a different chromosome. The normal genetic role of these elements is not known with
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An Introduction to Genetic Analysis
Chapter 20
Transposable Genetic Elements
certainty. They have been detected genetically through the abnormalities that they produce in
the activities and structures of the genes near the sites to which they move. A variety of
physical techniques have been used to detect them as well, including DNA sequencing.
Transposable genetic elements have been found in most organisms in which they have been
sought.
Today, transposable elements provide valuable tools both in prokaryotes and in eukaryotes for
genetic mapping, creating mutants, cloning genes, and even producing transgenic organisms.
Let us reconstruct some of the steps in the evolution of our present understanding of
transposable elements. In doing so, we will uncover the principles relevant to these
fascinating genetic units.
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 20-1). 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 20-2). 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 allele—an 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. When 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 outcome 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 can move, yielding reversion to A1. The A1 allele remains stable in the
absence of Dt.
McClintock's experiments: the Ds element
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An Introduction to Genetic Analysis
Chapter 20
Transposable Genetic Elements
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 20-3a) or genetically by the uncovering of recessive genes (Figure
20-3b). 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 20-3) 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 20-3b).
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 20-4), but one exceptional kernel
was very interesting. In Figure 20-4, 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 20-4 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, on 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.
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An Introduction to Genetic Analysis
Chapter 20
Transposable Genetic Elements
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 a1Dt 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 wxm−1/wxm−2 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
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An Introduction to Genetic Analysis
Chapter 20
Transposable Genetic Elements
likelihood an incomplete version of Ac itself.
Figure 20-5 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
20-6 and 20-7 show examples of the effects of transposons in maize and similar
effects in the snapdragon.
Bacterial insertion sequences
Insertion sequences, or insertion-sequence (IS) elements, are now known to be segments of
bacterial DNA that can move from one position on a chromosome to a different position on
the same chromosome or on a different chromosome. When IS elements appear in the middle
of genes, they interrupt the coding sequence and inactivate the expression of that gene. Owing
to their size and in some cases the presence of transcription and translation termination
signals, IS elements can also block the expression of other genes in the same operon if those
genes are downstream from the promoter of the operon. IS elements were first found in E. coli
in the gal operon—a set of three genes taking part in the metabolism of the sugar galactose.
Physical demonstration of DNA insertion
Recall that phage λ inserts next to the gal operon and that it is a simple matter to obtain λdgal
phage particles that have picked up the gal region (page 229). When the IS mutations in gal
are incorporated into λdgal phages and the buoyant density in a cesium chloride (CsCl)
gradient of the phages is compared with that of the normal λdgal phages, it is evident that the
DNA carrying the IS mutation is longer than the wild-type DNA. This experiment clearly
demonstrates that the mutations are caused by the insertion of a significant amount of DNA
into the gal operon. Figure 20-8 depicts this experiment in more detail.
Direct visualization of inserted DNA
When denatured λdgal DNA containing the insertion mutation is hybridized to denatured
wild-type λdgal DNA, the extra piece of DNA can be located under the electron microscope.
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An Introduction to Genetic Analysis
Chapter 20
Transposable Genetic Elements
In such experiments, some of the DNA molecules that form in the mixture are not the parent
duplexes but are heteroduplexes between one mutant and one wild-type strand. When point
mutations are analyzed, the heteroduplexes are indistinguishable from the parental DNA
molecules. However, in DNA containing the IS mutations, each heteroduplex shows a
single-stranded buckle, or loop (Figure 20-9). This single-stranded buckle confirms the
presence of an inserted sequence in the mutated DNA that has no complementary sequence in
the wild-type DNA. The length of this single-stranded loop can be calibrated by including
standardized marker DNA in the preparation. It proves to be approximately 800 nucleotides in
length.
Identification of discrete IS elements
Are the segments of DNA that insert into genes merely random DNA fragments or are they
distinct genetic entities? Hybridization experiments show that many different insertion
mutations are caused by a small set of insertion sequences. In these experiments, the λdgal
phages, which contain the gal− gene, are isolated from the IS mutant bacteria, and their DNA
is used to synthesize radioactive RNA in vitro. Certain fragments of this RNA are found to
hybridize with the mutant DNA but not with wild-type DNA, indicative of the fact that the
mutant contains an extra piece of DNA. These particular RNA fragments also hybridize to
DNA from other IS mutants, showing that the same bit of DNA is inserted in different places
in the different IS mutants.
On the basis of their patterns of cross-hybridization, the insertion mutants are placed into
categories. The first sequence, the 800-bp segment identified in gal, is termed IS1. A second
sequence, termed IS2, is 1350 bp long. Table 20-1 lists some of the insertion sequences and
their sizes. The inverted repeats listed in Table 20-1 will be dealt with shortly under
“Transposons.”
We now know that the genome of the standard wild-type E. coli is rich in IS elements: it
contains eight copies of IS1, five copies of IS2, and copies of other less well studied IS types.
It should be emphasized that the sudden appearance of an insertion sequence at any given
locus under study means that these elements are truly mobile, with a capability for
transposition throughout the genome. Presumably, they produce a mutation or some other
detectable alteration of normal cell function only when they happen to end up in an
“abnormal” position, such as the middle of a structural gene. Insertion sequences also are
commonly observed in the F factor. Figure 20-10 shows an example of an F-lac episome.
MESSAGE
The bacterial genome contains segments of DNA, termed IS elements, that can move
from one position on the chromosome to a different position on the same chromosome
or on a different chromosome.
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Transposable Genetic Elements
Orientation of IS elements
Because of the base sequence, the two strands of λdgal+ DNA happen to have different
buoyant densities. After DNA denaturation, they can be recovered separately in an
ultracentrifuge. In some cases, the same strands (that is, parallel strands) from two different
IS1 mutants form an unexpected hybrid with each other. Under the electron microscope, these
hybrids have a peculiar appearance: each is a double-stranded region with four
single-stranded tails (Figure 20-11). This observation is explained by assuming that the IS1
elements are inserted in opposite directions in the two mutants (Figure 20-12).
Prokaryotic transposons
A frightening ability of pathogenic bacteria was discovered in Japanese hospitals in the 1950s.
Bacterial dysentery is caused by bacteria of the genus Shigella. This bacterium initially
proved to be sensitive to a wide array of antibiotics that were used to control the disease. In
the Japanese hospitals, however, Shigella isolated from patients with dysentery proved to be
simultaneously resistant to many of these drugs, including penicillin, tetracycline,
sulfanilamide, streptomycin, and chloramphenicol. This multiple drugresistance phenotype
was inherited as a single genetic package, and it could be transmitted in an infectious
manner—not only to other sensitive Shigella strains, but also to other related species of
bacteria. This talent is an extraordinarily useful one for the pathogenic bacterium, and its
implications for medical science were terrifying. From the point of view of the geneticist,
however, the situation is very interesting. The vector carrying these resistances from one cell
to another proved to be a self-replicating element similar to the F factor. These R factors (for
resistance) are transferred rapidly on cell conjugation, much like the F particle in E. coli
(Chapter 7).
In fact, these R factors proved to be just the first of many similar F-like factors to be
discovered. These elements, which exist in the plasmid state in the cytoplasm, have been
found to carry many different kinds of genes in bacteria. Table 20-2 lists just some of the
characteristics that can be borne by plasmids. What is the mode of action of these plasmids?
How do they acquire their new genetic abilities? How do they carry them from cell to cell?
The discovery of transposons made it possible to answer some of these questions.
Physical structure of transposons
If the DNA of a plasmid conferring drug resistance (carrying the genes for kanamycin
resistance, for example) is denatured to single-stranded forms and then allowed to renature
slowly, some of the strands form an unusual shape under the electron microscope: a large,
circular DNA ring is attached to a “lollipop”-shaped structure (Figure 20-13). The “stick” of
the lollipop is double-stranded DNA, which has formed through the annealing of two
inverted repeat (IR) sequences in the plasmid (Figure 20-14). Subsequent studies have
shown that the IR sequences are a pair of IS elements in many cases. For instance, IS10 is
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An Introduction to Genetic Analysis
Chapter 20
Transposable Genetic Elements
present at the ends of the region carrying the genes for tetracycline resistance (Figure 20-15).
In some cases, however, the IR sequences are much smaller.
The genes for drug resistance or other genetic abilities carried by the plasmid are located
between the IR sequences in the lollipop head. The IR sequences together with their contained
genes have been collectively called a transposon (Tn). (Transposons are therefore longer
than IS elements, because they contain extra protein-encoding genes.) The remainder of the
plasmid, bearing the genes encoding resistance-transfer functions (RTF), is called the RTF
region (Figure 20-16). Table 20-3 lists some of the known transposons.
Movement of transposons
A transposon can jump from a plasmid to a bacterial chromosome or from one plasmid to
another plasmid. In this manner, multiple drug-resistant plasmids are generated. Figure 20-17
is a composite diagram of an R plasmid, indicating the various places at which transposons
can be located.
MESSAGE
Transposons were originally detected as mobile genetic elements that confer drug
resistance. Many of these elements consist of recognizable IS elements flanking a
gene that encodes drug resistance. IS elements and transposons are now grouped
together under the single term transposable elements.
Phage mu
Phage mu is a normal-appearing phage. We consider it here because, although it is a true virus,
it has many features in common with IS elements. The DNA double helix of this phage is
36,000 nucleotides long—much larger than an IS element. However, it does appear to be able
to insert itself anywhere in a bacterial or plasmid genome in either orientation. Once inserted,
it causes mutation at the locus of insertion—again like an IS element. (The phage was named
for this ability: mu stands for “mutator.”) Normally, these mutations cannot be reverted, but
reversion can be produced by certain kinds of genetic manipulation. When this reversion is
produced, the phages that can be recovered show no deletion, proving that excision is exact
and that the insertion of the phage therefore does not involve any loss of phage material
either.
Each mature phage particle has on each end a piece of flanking DNA from its previous host
(Figure 20-18). However, this DNA is not inserted anew into the next host. Its function is
unclear. Phage mu also has an IR sequence, but neither of the repeated elements is at a
terminus.
Mu can also act like a genetic snap fastener, mobilizing any kind of DNA and transposing it
anywhere in a genome. For example, it can mobilize another phage (such as λ) or the F factor.
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An Introduction to Genetic Analysis
Chapter 20
Transposable Genetic Elements
In such situations, the inserted DNA is flanked by two mu genomes (Figure 20-19). It can also
transfer bacterial markers onto a plasmid; here again, the transferred region is flanked by a
pair of mu genomes (Figure 20-20). Finally, the phage mu can mediate various kinds of
structural chromosome rearrangements (Figure 20-21).
Mechanism of transposition in prokaryotes
Several different mechanisms of transposition are employed by prokaryotic transposable
elements. And, as we shall see later, eukaryotic elements exhibit still additional mechanisms
of transposition.
In E. coli, we can identify replicative and conservative (nonreplicative) modes of
transposition. In the replicative pathway, a new copy of the transposable element is generated
in the transposition event. The results of the transposition are that one copy appears at the new
site and one copy remains at the old site. In the conservative pathway, there is no replication.
Instead, the element is excised from the chromosome or plasmid and is integrated into the
new site.
Replicative transposition
When transposition is from one locus to a second locus for certain transposons, a copy of the
transposable element is left behind at the first locus. An analysis of transposon mutants
revealed an interesting fact about the mechanism of transposition. Using the transposon Tn3
(Figure 20-22), researchers grouped the mutations that prevent transposition into two
categories. A trans-recessive class maps in the gene that encodes the transposase enzyme, a
catalyst of transposition. A second class of cis-dominant mutations results in the buildup of an
intermediate in the transposition process. Figure 20-23 diagrams the transposition pathway in
the Tn3 transposition from one plasmid to another. The intermediate is a double plasmid, with
both donor and recipient plasmid being fused together. The combined circle resulting from the
fusion of two circular elements is termed a cointegrate. Apparently, the mutations in this
second class delete a region on the transposon at which a recombination event takes place that
resolves cointegrates into two smaller circles. This region, called the internal resolution site
(IRS), appears in Figure 20-22.
The finding of a cointegrate structure as an intermediate in transposition helped establish a
replicative mode of transposition for certain elements. In Figure 20-23, note how the
transposable element is duplicated in the fusion event and how the recombination event that
resolves the cointegrate into two smaller circles leaves one copy of the transposable element
in each plasmid.
Conservative transposition
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Chapter 20
Transposable Genetic Elements
Some transposons, such as Tn10, excise from the chromosome and integrate into the target
DNA. In these cases, DNA replication of the element does not occur, and the element is lost
from the site of the original chromosome. Researchers demonstrated this lack of replication
by constructing heteroduplexes of λTn10 derivatives containing the lac region of E. coli. The
researchers used DNA from Tn10-lacZ+ and Tn10-lacZ− derivatives. The heteroduplexes,
therefore, contain one strand with the wild-type lac region and a second strand with the
mutated (Z−) lac region. Figure 20-24 diagrams this part of the experiment. The heteroduplex
DNA is used to infect cells that have no lac genes, and transpositions of the TetR Tn10 are
selected. Different types of colonies arise from the transposition of a heteroduplex Z−/Z+
carrying transposon (Figure 20-25). If replication takes place (the replicative mode of
transposition), all colonies are either completely Lac+ or completely Lac−, because the
replication will convert the heteroduplex DNA into two homoduplex daughter molecules. The
mechanism by which this conversion takes place will be examined in detail in the next
section. However, if the transposition is conservative and does not include replication, each
colony arises from a lacZ+/lacZ− heteroduplex. Such colonies are partly Lac+ and partly Lac−.
By using media that stain Lac+ and Lac− cells different colors, researchers can observe the
Lac+ and Lac− sectors in colonies.
Therefore, the determination of whether Tn10 undergoes replicative or conservative
transposition can be made by observing whether differently colored sectors exist within the
same colony resulting from the transposition. Sectored colonies are observed in a majority of
cases (Figure 20-26). Thus, Tn10—and perhaps other transposable elements in E.
coli—transpose by excising themselves from the donor DNA and integrating directly into the
recipient DNA.
Molecular consequences of transposition
The molecular consequences of transposition reveal an additional piece of evidence
concerning the mechanism of transposition: on integration into a new target site, transposable
elements generate a repeated sequence of the target DNA in both replicative and conservative
transposition. Figure 20-27 depicts the integration of IS1 into a gene. In the example shown,
the integration event results in the repetition of a 9-bp target sequence. Analysis of many
integration events reveals that the repeated sequence does not result from reciprocal
site-specific recombination (as is the case in phage λ integration; see page 229); rather, it is
generated in the process of integration itself. The number of base pairs is a characteristic of
each element. In bacteria, 9-bp and 5-bp repeats are most common.
The preceding observations have been incorporated into somewhat complicated models of
transposition. Most models postulate that staggered cleavages are made at the target site and
at the ends of the transposable element by a transposase enzyme that is encoded by the
element. One end of the transposable element is then attached by a single strand to each
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Transposable Genetic Elements
protruding end of the staggered cut. Subsequent steps depend on the mode of transposition
(replicative or conservative).
Rearrangements mediated by transposable elements
Transposable elements generate a high incidence of deletions in their vicinity. These deletions
emanate from one end of the element into the surrounding DNA (Figure 20-28). Such events,
as well as element-induced inversions, can be viewed as aberrant transposition events.
Transposons also give rise to readily detectable deletions in which part of the element is
deleted together with varying lengths of the surrounding DNA. This process of imprecise
excision is now recognized as deletions or inversions emanating from the internal ends of the
IR segments of the transposon. The process of precise excision—the loss of the transposable
element and the restoration of the gene that was disrupted by the insertion—also occurs,
although at very low rates compared with the frequencies of the events just described.
MESSAGE
Some DNA sequences in bacteria and phages act as mobile genetic elements. They
are capable of joining different pieces of DNA and are thus capable of splicing DNA
fragments into or out of the middle of a DNA molecule. Some naturally occurring
mobile or transposable elements carry antibiotic-resistance genes.
Review of transposable elements in prokaryotes
Let's examine what we have learned so far about prokaryotic transposable elements:
1. There are several different types of transposable elements, including insertion sequences
(IS1, IS2, . . . ) and transposons (Tn1, Tn2, . . . ).
2. Two copies of a transposable element can act in concert to transpose the DNA segments in
between them. Some of the transposons that confer antibiotic resistance are formed in this
manner, with two insertion sequences flanking the genes for antibiotic resistance.
3. Most of the transposable elements have recognizable inverted repeat (IR) structures, some
of which can be observed under the electron microscope after denaturation and renaturation.
4. Transposable elements are found in bacterial chromosomes, as well as in plasmids.
5. After insertion into a new site on the DNA, transposable elements generate a short repeated
sequence, commonly consisting of 9 or 5 bp.
6. The detailed mechanism of transposition is not known, but two different pathways for
transposition have been identified. In some cases, transposition takes place by replication of a
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An Introduction to Genetic Analysis
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Transposable Genetic Elements
new copy of the element into the target site, with one copy being left behind at the original
site. In other cases, transposition consists of the excision of the element from the original site
and its reintegration into a new site. These two modes of transposition are called replicative
and conservative, respectively.
Transposable elements have been found in eukaryotes, and some have close similarities to
those observed in bacteria, transposing through DNA intermediates. Interestingly, other
mobile elements transpose through RNA intermediates and, in certain cases, resemble
mammalian retroviruses.
Molecular nature of transposable elements in eukaryotes
Transposable genetic elements are even more prevalent in eukaryotic chromosomes than in
bacterial chromosomes. For instance, from 25 percent to 40 percent of mammalian
chromosomes consist of transposable elements that have accumulated in the course of
evolution. In addition, half of the spontaneous mutations seen in Drosophila are attributed to
the movement and insertion of transposable elements. The phenotype conferred by the
transposed element was used to detect the first elements in maize, as well as in bacteria—for
instance, the patched kernels in corn and the Gal − mutants in bacteria. Now, most elements
are detected by recognizing their characteristic structures after sequencing large chromosomal
regions. Some eukaryotic elements, such as the maize Ac and Ds elements, and the
Drosophila P elements discussed in a later section, transpose as DNA, as do all prokaryotic
elements. However, most eukaryotic elements use a different mechanism, transposing in the
same manner as that of RNA viruses. Because many transposable elements appear to be
related to single-stranded RNA animal viruses, we shall examine some aspects of these
viruses.
Retroviruses
Retroviruses are single-stranded RNA animal viruses that employ a double-stranded DNA
intermediate for replication. The RNA is copied into DNA by the enzyme reverse
transcriptase. The life cycle of a typical retrovirus is shown in Figure 20-29. Some
retroviruses, such as mouse mammary tumor virus (MMTV) and Rous sarcoma virus (RSV),
are responsible for the induction of cancerous tumors. When integrated into host
chromosomes as double-stranded DNA, these retroviruses are termed proviruses. Proviruses,
like the mu phage in bacteria, can be considered transposable elements, because they can in
effect transpose from one location to another. Figure 20-30 depicts transposition by a
retroviral mechanism.
Retroviruses are structurally similar to transposable elements in other organisms, as we shall
see later. Additionally, integration results in the duplication of a short target sequence in the
host chromosome.
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Retrotransposons
Transposable elements that utilize reverse transcriptase to transpose through an RNA
intermediate are termed retrotransposons. They are widespread among eukaryotes and are
generally divided into two classes. Viral retrotransposons have properties similar to those of
retroviruses. For instance, they display long terminal repeats, or LTRs, as shown in Figure
20-31. Two examples of viral retrotransposons are the yeast Ty elements, and the
Drosophilacopia elements.
Figure 20-32 shows the structure of one of the Ty elements in yeast: the Ty1 sequence, which
is present in approximately 35 copies in the yeast genome. The 330-bp-long termini (terminal
sequences), called δ (delta) sequences, are present in about 100 copies in the genome. Yeast δ
sequences, as well as Ty elements as a whole, show significant sequence divergence. Ty
elements generate a repeated sequence of target DNA (in this case, 5 bp) during transposition,
like prokaryotic transposons. Ty elements cause mutations by insertion into different genes in
the yeast chromosome. In 1985, Jef Boeke and Gerald Fink use Ty1 to show that the
transposition of Ty elements is through an RNA intermediate.
Figure 20-33 diagrams the experimental design used by Boeke and Fink and their colleagues
to alter a yeast Ty element, cloned on a plasmid. First, a promoter was inserted near the end of
an element that could be activated by the addition of galactose to the medium. The use of a
galactose-sensitive promoter allows the manipulation of the expression of Ty RNA. Galactose
enhances the transcription of Ty RNA. Second, an intron from another yeast gene was
introduced 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 involvement of RNA, because
galactose-stimulated transcription begins at the galactose-sensitive promoter (Figure 20-33).
The key experimental result, however, is the fate of the transposed Ty DNA. When the
researchers examined the Ty DNA resulting from transpositions, they found that the intron
had been removed. Because introns are excised only in the course of RNA processing (see
Chapter 10), the transposed Ty DNA must have been copied from an RNA intermediate
transcribed from the original Ty element and then processed by RNA–RNA splicing. The
DNA copy of the spliced mRNA is then integrated into the yeast chromosome.
The copia-like elements of Drosophila constitute at least seven families, ranging in size from
5 kb to 8.5 kb. Members of each family appear at 10–100 positions in the Drosophila genome.
Each member carries a long, direct terminal repeat and a short, imperfect inverted repeat
(Figure 20-34) and is structurally similar to a yeast Ty element. Copia-like elements also
cause a duplication of a characteristic number of base pairs of Drosophila DNA on insertion.
Certain classic Drosophila mutations result from the insertion of copia-like and other
elements. For example, the white-apricot (wa) mutation for eye color is caused by the insertion
of an element from the copia family into the white locus.
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An Introduction to Genetic Analysis
Chapter 20
Transposable Genetic Elements
Figure 20-35 shows the similarities in the arrangements of genes between integrated
retrovirus DNA and the Ty and copia elements. Note how each of the proteins encoded by the
retrovirus have counterparts in Ty and copia transposons.
Nonviral retrotransposons: LINES and SINES
Nonviral retrotransposons are the most frequently encountered transposons in mammals.
The LINEs (long interspersed elements) and SINEs (short interspersed elements; including
the Alu sequences) discussed in Chapter 3 are the two most abundant. All of these elements
are repeated many times in mammalian genomes. The human genome has from 20,000 to
40,0000 LINE elements and about 500,000 SINE elements, most of which display some
sequence divergence. Some idea of the profusion of repetitive elements in the human genome
can be obtained from Figure 20-36, which shows the distribution of repetitive elements in the
gene for homogentisate 1,2-dioxygenase [the alkaptonuria gene (AKU or HGO)].
Function of transposable elements
Transposable elements play a role in the biology of organisms. They cause mutations by
insertion into genes and affect the regulation of genes by inserting near promoters. They also
provide substrates for genetic rearrangements and thus act as agents of genome evolution. For
instance, in E. coli growing in natural environments, a sizable fraction of spontaneous
mutations are caused by the IS series of transposable elements. As mentioned earlier, more
than half of the spontaneous mutations in Drosophila result from transposable-element
insertion. Insertions of IS elements near the promoter region of the bgl operon, which encodes
proteins participating in β-glucosidase metabolism, activates transcription in this normally
“cryptic” operon. There are numerous other examples of insertions that turn on gene
expression.
Insertion elements provide portable regions of homology that can serve as substrates for
recombination enzymes, creating deletions, duplications, and inversions. There is evidence
that transposable element-mediated rearrangements may have played an important role in
generating the genomes of different organisms, as well as contributing to new functions by
stimulating gene duplication. Transposable elements have accumulated during evolution to
the point where they constitute much of the genome of higher cells. For instance, in humans,
approximately 30 percent of the genomic DNA consists of transposable elements. The
evolution of different antibiotic-resistance-carrying microorganisms is influenced by
transposable elements. Recall the transposons that can generate different combinations of
resistance-encoding genes on R plasmids that are then transferred to different bacteria.
Uses of transposable elements
Transposable elements have many uses. In prokaryotes, the antibiotic-resistance marker
carried by different transposons serves as a convenient marker. For instance, derivatives of
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Tn10 are often used to create insertions in the bacterial chromosome. After selection for
tetracycline-resistant (Tetr) cells, each of which contains an insertion somewhere in the gene,
the position of the insert can be mapped genetically, by following the Tetr phenotype, or
physically, by using primers matching the ends of Tn10 and then matching the sequence of
the adjacent bacterial DNA with that from the known sequence of the entire genome (in S.
typhimurium and E. coli for example). With the use of linked transposons, genes can be
transferred from one strain to another easily and can be cloned by selecting for the
antibioticresistance marker that is either inserted into or very near the gene of interest. In
eukaryotes, transposable elements are also used for generating insertion mutations, mapping
them, and facilitating both the cloning of genes and the generation of transgenic
organisms.
P elements
The P element in Drosophila is one of the best examples of exploiting the properties of
transposable elements in eukaryotes. This element, shown in Figure 20-37, is 2907 bp long
and features a 31-bp inverted repeat at each end. The element encodes a transposase.
Although the transposase is required for transposition, it can be supplied by a second element.
Therefore, P elements with internal deletions can be mobilized and then remain fixed in the
new position in the absence of the second element; thus the P element can serve as a
convenient marker. P elements do not utilize an RNA intermediate during transposition and
can insert at many different positions in the Drosophila chromosome. The transposition of a P
element is controlled by repressors encoded by the element.
P elements have been developed as tools for Drosophila much in the same way that
transposons such as Tn10 have for bacteria. Namely, 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 can be inserted into genes in vivo, and different phenotypes can be selected. Then,
the interrupted gene can be cloned, with the use of P element segments as a probe, a method
termed transposon tagging. Primers matching the 31-bp sequence at each end can be used to
sequence chromosomal regions adjacent to P element insertion sites.
Using P elements to insert genes
Gerald Rubin and Allan Spradling showed that P element DNA can be used as an effective
vehicle for transferring donor genes into the germ line of a recipient fly. Rubin and Spradling
devised the following experimental procedure (Figure 20-38). The recipient genotype is
homozygous for the rosy (ry−) mutation, which confers a characteristic eye color. 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 deleted P element
into which the ry+ gene has been spliced. This deleted element is not able to transpose, owing
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to the deletion; so, as mentioned earlier, a helper plasmid bearing a complete element also is
injected. Flies developing from these embryos are phenotypically still rosy mutants, but their
offspring include a large proportion of ry+ flies. These ry+ descendants show Mendelian
inheritance of the newly acquired ry+ gene, suggesting that it is located on a chromosome.
This location was confirmed by in situ hybridization, which shows that the ry+ gene, together
with the deleted P element, has been inserted into one of several distinct chromosome
locations. None appears exactly at the normal locus of the rosy gene. These new ry+ genes are
found to be inherited in a stable fashion. A variation of this method, described in Chapter 13,
uses P elements to make transgenic Drosophila by transferring foreign genes into the germ
line and then monitoring their expression pattern.
Review of transposable elements in eukaryotes
Let's examine some of the essential points about eukaryotic transposable elements:
1. Transposable elements exist in all cells. Elements in yeast, Drosophila, and maize have
been well studied, as have retroviruses in mammalian cells.
2. Some transposable elements can be used as tools for cloning and gene manipulation. For
instance, the P elements of Drosophila can be employed to transfer genes into the germ line
of a recipient fly. As another example, the T-DNA segment of Ti plasmids (described in
Chapter 13) can be used to introduce cloned genes into certain plants.
3. A similarity between eukaryotic transposable elements and their counterparts in
prokaryotes is that transposition into a new site generates a short repeated sequence at the
target site.
4. A difference between certain eukaryotic and prokaryotic transposable elements lies in the
mechanism of transposition. Some eukaryotic transposable elements transpose through an
RNA intermediate; prokaryotic elements do not use an RNA intermediate.
Summary
Nature has devised many different ways of changing the genetic architecture of organisms.
We are now beginning to understand the molecular processes behind some of these
phenomena. Gene mutation, recombination between chromosomes, and transposition can all
be reasonably explained at the DNA level. Far from merely producing genetic waste, all these
processes undoubtedly have important roles in evolution. This idea is strengthened through
the knowledge that the processes themselves are to a large extent under genetic control: there
are genes that affect the efficiency of mutation, recombination, and transposition.
Although different mechanisms of transposition are sometimes used, the analogies between
the transposable elements of phages, bacteria, and eukaryotes are striking. At present, it is not
known if transposons are elements that normally play a role in the day-to-day transactions of
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the genome, as originally proposed by Barbara McClintock in the 1950s, or if they are pieces
of “selfish DNA” that exist for no purpose other than their own survival. Whatever the truth
of this matter is, transposons certainly constitute a completely unexpected element of chaos in
the genome, which geneticists have already harnessed into their team of analytical procedures.
At the evolutionary level, transposons may be important in the sudden leaps that characterize
the fossil record.
Problems
1. Suppose that you want to determine whether a new mutation in the gal region of E. coli is
the result of an insertion of DNA. Describe two physical experiments that would allow you
to demonstrate the presence of an insertion.
2. Explain the difference between the replicative and conservative modes of transposition.
Briefly describe an experiment demonstrating each of these modes in prokaryotes.
3. Describe the generation of multiple drug-resistance plasmids.
See answer
4. Briefly describe the experiment that demonstrates that the transposition of the Ty1 element
in yeast takes place through an RNA intermediate.
See answer
5. Explain how the properties of P elements in Drosophila make possible gene-transfer
experiments in this organism.
See answer
6. When Rhoades took pollen from wholly pigmented anthers on plants of genotype a1/a1 ;
Dt/Dt and used this pollen to pollinate a1/a1 ; dt/dt tested females, he found wholly
pigmented kernels and, in addition, some dotted kernels. Explain the origin of both
phenotypes.
7. In Drosophila, M. Green found a singed allele (sn) with some unusual characteristics.
Females homozygous for this X-linked allele have singed bristles, but they have numerous
patches of sn+ (wild-type) bristles on their heads, thoraxes, and abdomens. When these flies
are mated with sn males, some females give only singed progeny, but others give both
singed and wild-type progeny in variable proportions. Explain these results.
See answer
8. Crown gall tumors are found in many dicotyledonous plants infected by the bacterium
Agrobacterium tumefaciens. The tumors are caused by the insertion of DNA from a large
plasmid carried by the bacterium into the plant DNA. Suppose that a tobacco plant of type
A (there are many types of tobacco plants) is infected, and it produces tumors. You remove
tumor tissue and grow it on a synthetic medium. Some of these tumor cultures produce
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An Introduction to Genetic Analysis
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aerial shoots. You graft one of these shoots onto a normal tobacco plant of type B, and the
graft grows to an apparently normal A-type shoot and flowers.
a. You remove cells from the graft and place them in synthetic medium, where they grow
like tumor cells. Explain why the graft appears to be normal.
b. When seeds are produced by the graft, the resulting progeny are normal A-type plants.
No trace of the inserted plasmid DNA remains. Propose a possible explanation for this
“reversal.”
See answer
9. Consider two maize plants:
a. Genotype C/cm ; Ac/Ac+ where cm is an unstable allele caused by Ds insertion
b. Genotype C/cm, where cm is an unstable allele caused by Ac insertion
What phenotypes would be produced and in what proportions when (1) each plant is
crossed with a base-pair-substitution mutant c/c and when (2) the plant in part a is crossed
with the plant in part b? Assume that Ac and c are unlinked, that the chromosome breakage
frequency is negligible, and that mutant c/C is Ac+.
Chapter 20*
3. R plasmids are the main carriers of drug resistance. They acquire these genes by
transposition of drug-resistance genes located between IR (inverted repeat) sequences.
Once in a plasmid, the transposon carrying drug resistance can be transferred on
conjugation if it stays in the R plasmid or it can insert into the host chromosome.
4. Boeke, Fink, and their co-workers demonstrated that transposition of the Ty element in
yeast requires an RNA intermediate. They constructed a plasmid by using a Ty element that
had a promoter that could be activated by galactose and an intron inserted into its coding
region. First, the frequency of transposition was greatly increased by the addition of
galactose, indicating that an increase in transcription (and production of RNA) was
correlated to rates of transposition. More importantly, after transposition, they found that
the newly transposed Ty DNA lacked the intron sequence. Because intron splicing takes
place only in RNA processing, there must have been an RNA intermediate in the
transposition event.
5. P elements are transposable elements found in Drosophila. Under certain conditions they
are highly mobile and can be used to generate new mutations by random insertion and gene
knockout. As such, they are a valuable tool to tag and then clone any number of genes. (See
Problem 15 from Chapter 13 for a discussion on cloning by tagging.) P elements can also
be manipulated and used to insert almost any DNA (or gene) into the Drosophila genome.
P element–mediated gene transfer works by inserting the DNA of interest between the
inverted repeats necessary for P element transposition and injecting this recombinant DNA
along with helper intact P element DNA (to supply the transposase) into very early
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An Introduction to Genetic Analysis
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embryos and screening for (random) insertion among the injected fly's offspring.
7. The best explanation is that the mutation is due to an insertion of a transposable element.
8. a. and b. The soil bacterium Agrobacterium tumefaciens contains a large plasmid called the
Ti (tumor-inducing) plasmid. When this bacterium infects a plant, a region of the Ti
plasmid called the T-DNA is transferred and inserted randomly into the plant's genome.
The T-DNA directs the synthesis of plant hormones that cause uncontrolled growth (a
tumor) and also directs the plant's synthesis of compounds called opines. (These
compounds cannot be metabolized by the plant but are used by the bacterium.)
When a piece of “normal” plant tissue is cultured with appropriate nutrients and growth
hormones, cells are stimulated to divide in a disorganized manner, forming a mass of
undifferentiated cells called a callus. These cells will differentiate only into shoots (or
roots) if the levels of growth hormones are carefully adjusted. The T-DNA causes
undifferentiated growth because it directs the unbalanced synthesis of these same
hormones. The fact that some of the infected cultures produced shoots suggests that these
cells “lost” the ability to overproduce these hormones. This would be consistent with the
loss of the T-DNA (similar to the loss of other transposable elements that is observed in
many species). Thus, the A graft would grow normally, and seeds produced by the graft
would have no trace of the T-DNA. The fact that cells from the A graft grow like tumor
cells when placed on synthetic medium suggests that the medium supplies the high levels
of hormones necessary for undifferentiated growth even in the absence of T-DNA.
勇者并非无所畏惧, 而是判断出有比恐惧更重要的东西.