Chapter 6 Transposable elements

 Transposable elements, also known as transposons or “jumping genes,” can move or transpose within and
between chromosomes, inserting themselves into various locations within the genome.
 Transposons are present in the genomes of all organisms from bacteria to humans. Not only are they
ubiquitous, but they also comprise large portions of some eukaryotic genomes. For example, almost 50
percent of the human genome is derived from transposable elements. Some organisms with unusually large
genomes, such as salamanders and barley, contain hundreds of thousands of copies of various types of
transposable elements.
 Although the function of these elements is unknown, data from human genome sequencing suggest that
some genes may have evolved from transposons and that transposons may help to modify and reshape the
genome.
 Transposable elements fall into two general classes based on how they move from location to location in the
genome.
 One class—found in both prokaryotes and eukaryotes—moves as a DNA segment.
 Members of the other class—found only in eukaryotes—are related to retroviruses and move via an RNA.
First an RNA copy of the element is synthesized; then a DNA copy of that RNA is made, and it integrates at a
new site in the genome.
 In bacteria, transposable elements can move to new positions on the same chromosome (because there is
only one chromosome) or onto plasmids or phage chromosomes;
 In eukaryotes, transposable elements may move to new positions within the same chromosome or to a
different chromosome. In both bacteria and eukaryotes, transposable elements insert into new chromosome
locations with which they have no sequence homology; therefore, transposition is a process different from
homologous recombination (recombination between matching DNA sequences) and is called
nonhomologous recombination.
 Transposable elements are important due to the genetic changes they cause. For example, they can produce
mutations by inserting into genes (a process called insertional mutagenesis), they can increase or decrease
gene expression by inserting into gene regulatory sequences (such as by disrupting promoter function or
stimulating a gene’s expression through the activity of promoters on the element), and they can produce
various kinds of chromosomal mutations through the mechanics of transposition. In fact, transposable
elements have made important contributions to the evolution of the genomes of both bacteria and
eukaryotes through the chromosome rearrangements they have caused. The frequency of transposition,
though typically low, varies with the particular element. If the frequency were high, the genetic changes
caused by the transpositions would likely kill the cell

Uses of transposons-
o Valuable tool in genetic research.
o Used as cloning vectors
o Transformation vectors for transferring genes between organisms
o Drug resistance genes encoded by many transposons used in the development of plasmids as
cloning vectors
o Transposons used to increase the rate of mutation due to insertional inactivation
o Especially useful tool in plant molecular biology
o Used as a means to identify mutant allele
o Used to study chemical mutagenesis methods
o Used to study gene expression
o Used as a tool of mutagenesis of most experimentally tractable organisms.
Eukaryotic transposable elements
1. Ty Transposable Elements in Yeast
 A Ty transposable element is about 5.9 kb long and includes two directly repeated
terminal sequences called long terminal repeats (LTR) or deltas ( ).
 Each delta contains a promoter and sequences recognized by transposing enzymes. The Ty
elements encode a single, 5,700-nucleotide mRNA that begins at the promoter in the
delta at the left end of the element .
 The mRNA transcript contains two open reading frames (ORFs), designated TyA and TyB,
that encode two different proteins required for transposition. On average, a strain contains
about 35 Ty elements.
 Ty elements are similar to retroviruses—singlestranded RNA viruses that replicate via
double-stranded DNA intermediates. That is, when a retrovirus infects a cell, its RNA
genome is copied by reverse transcriptase(RNA- dependent DNA polymerase), an enzyme
that enters the cell as part of the virus particle. meaning that the enzyme uses an RNA
template to produce a DNA copy. The enzyme then catalyzes the synthesis of a
complementary DNA strand, in the end producing a double-stranded DNA copy of the RNA
genome. The DNA integrates into the host’s chromosome, where it can be transcribed to
produce progeny RNA viral genomes and mRNAs for viral proteins. As a result of their
similarity to retroviruses,
 Ty elements were hypothesized to transpose not by a DNA-to-DNA mechanism, but
by making an RNA copy of the integrated DNA sequence and then creating a new Ty
element by reverse transcription.
 The new element would then integrate at a new chromosome location.
 Evidence substantiating the hypothesis was obtained through experiments with Ty elements
modified by DNA manipulation techniques to have special features enabling their
transposition to be monitored easily. One compelling piece of evidence came from
experiments in which an intron was placed into the Ty element (there are no introns in
normal Ty elements) and the element was monitored from its initial placement through the
transposition event. At the new location, the Ty element no longer had the intron sequence.
This result could only be interpreted to mean that transposition occurred via an RNA
intermediate.
 Subsequently, it was shown that Ty elements encode a reverse transcriptase. Moreover, Ty
virus like particles containing Ty RNA and reverse transcriptase activity have been identified
in yeast cells. Because of their similarity to retroviruses in this regard, Ty elements are
called retrotransposons, and the transposition process is called retrotransposition
 2. Copia and P Elements in Drosophila
 Copia element
 There are more than 30 families of transposable elements in Drosophila, each of which is present in 20
to 50 copies in the genome. Together, these families constitute about 5 percent of the Drosophila
genome and over half of the middle repetitive DNA of this organism. One study suggests that 50
percent of all visible mutations in Drosophila are the result of the insertion of transposons into
otherwise wild-type genes.
 In 1975, David Hogness and his colleagues David Finnegan, Gerald Rubin, and Michael Young
identified a class of DNA elements in Drosophila melanogaster that they designated as copia.
 These elements are transcribed into “ copious” amounts of RNA (hence their name). Copia elements
are present in 10 to 100 copies in the genomes of Drosophila cells.
 Mapping studies show that they are transposable to different chromosomal locations and are
dispersed throughout the genome
 Each copia element consists of approximately 5000 to 8000 bp of DNA, including a long
direct terminal repeat (DTR) sequence of 267 bp at each end. Within each DTR is an inverted
terminal repeat (ITR) of 17 bp. The short ITR sequences are characteristic of copia elements
 Insertion of copia is dependent on the presence of the ITR sequences and seems to occur
preferentially at specific target sites in the genome. The copia-like elements demonstrate regulatory
effects at the point of their insertion in the chromosome.
 Certain mutations, including those affecting eye color and segment formation, are due to copia
insertions within genes. For example, the eye-color mutation whiteapricot, is caused by an allele of the
white gene, which contains a copia element within the gene. Transposition of the copia element out of
the white-apricot allele can restore the allele to wild type.
 P element
 Perhaps the most significant Drosophila transposable elements are the P elements. These were
discovered while studying the phenomenon of hybrid dysgenesis, a condition characterized by sterility,
elevated mutation rates, and chromosome rearrangements in the offspring of crosses between certain
strains of fruit flies.
 Hybrid dysgenesis is caused by high rates of P element transposition in the germ line, in which
transposons insert themselves into or near genes, thereby causing mutations.
 P elements range from 0.5 to 2.9 kb long, with 31-bp ITRs. Full-length P elements encode at least
two proteins, one of which is the transposase enzyme that is required for transposition, and
another is a repressor protein that inhibits transposition.
 The transposase gene is expressed only in the germ line, accounting for the tissue specificity of P
element transposition. Strains of flies that contain full-length P elements inserted into their genomes
are resistant to further transpositions due to the presence of the repressor protein encoded by the P
elements.
 Mutations can arise from several kinds of insertional events. If a P element inserts into the coding
region of a gene, it can terminate transcription of the gene and destroy normal gene expression. If it
inserts into the promoter region of a gene, it can affect the level of expression of the gene. Insertions
into introns can affect splicing or cause the premature termination of transcription.
 Geneticists have harnessed P elements as tools for genetic analysis. One of the most useful
applications of P elements is as vectors to introduce transgenes into Drosophila—a technique
known as germ-line transformation. P elements are also used to generate mutations and to clone
mutant genes. In addition, researchers are perfecting methods to target P element insertions to precise
single-chromosomal sites, which should increase the precision of germ-line transformation in the
analysis of gene activity.
3. The Ac-Ds Transposable Elements in Corn.
Discovered by Barbara McClintock
About 20 years before the discovery of transposons in bacteria, Barbara McClintock discovered mobile
genetic elements in corn plants (maize). She did this by analyzing the genetic behavior of two mutations,
Dissociation (Ds) and Activator (Ac), expressed in either the endosperm or aleurone layers. She then
correlated her genetic observations with cytological examinations of the maize chromosomes. Initially,
McClintock determined that Ds was located on chromosome 9. If Ac was also present in the genome, Ds
induced breakage at a point on the chromosome adjacent to its own location. If chromosome breakage
occurred in somatic cells during their development, progeny cells often lost part of the broken chromosome,
causing a variety of phenotypic effects.
.. The Ac-Ds family of controlling elements has been studied in detail. The autonomous Ac element is 4,563
bp long, with short terminal inverted repeats and a single gene encoding the transposase.
.. Upon insertion into the genome, it generates an 8-bp direct duplication of the target site.
.. Ds elements are heterogeneous in length and sequence, but all have the same terminal IRs as Ac elements,
because most have been generated from Ac by the deletion of segments or by more complex sequence
rearrangements. As a result, Ds elements have no complete transposase gene; hence, these elements
cannot transpose on their own. Transposition of the Ac element occurs only during chromosome replication
and is a result of the cut-and-paste (conservative) transposition mechanism.
.. Consider a chromosome with one copy of Ac at a site called the donor site. When the chromosome region
containing Ac replicates, two copies of Ac result, one on each progeny chromatid.
..There are two possible results of Ac transposition, depending on whether it occurs to a replicated or an
unreplicated chromosome site.
.. If one of the two Ac elements transposes to a replicated chromosome site, an empty donor site is left on
one chromatid, and an Ac element remains in the homologous donor site on the other chromatid. The
transposing Ac element inserts into a new, already replicated recipient site, which is often on the same
chromosome.. Thus, in the case of transposition to an already replicated site, there is no net increase in the
number of Ac elements..
As in the first case, one of the two Ac elements transposes, leaving an empty donor site on one chromatid
and an Ac element in the homologous donor site on the other chromatid. But now the transposing element
inserts into a nearby recipient site that has yet to be replicated. When that region of the chromosome
replicates, the result will be a copy of the transposed Ac element on both chromatids, in addition to the one
original copy of the Ac element at the donor site on one chromatid. Thus, in the case of transposition to an
unreplicated recipient site, there is a net increase in the number of Ac elements.
.. The transposition of most Ds elements occurs in the same way as Ac transposition, using transposase
supplied by an Ac element in the genome. The first Ds element studied (Ds9) is nearly identical to Ac except
for a 194-bp deletion within the transposase gene. The deletion of part of the transposase gene in the Ds9
element explains its dependence on the Ac element for transposition. Several other Ds elements have also
been sequenced, and each contains an even larger deletion within the transposase gene. In each case,
however, the ITRs are retained
Transposable Elements in Bacteria
Two examples of transposable elements in bacteria are insertion sequence (IS) elements and transposons (Tn).

 Insertion Sequences. An insertion sequence (IS), or IS element, is the simplest transposable element found in
bacteria.
 An IS element contains only genes required to mobilize the element and insert it into a new location in the
genome. IS elements are normal constituents of bacterial chromosomes and plasmids
 IS elements were first identified in E. coli as a result of their effects on the expression of three genes that control
the metabolism of the sugar galactose. Some mutations affecting the expression of these genes did not have
properties typical of point mutations or deletions, but rather had an insertion of an approximately 800-bp DNA
segment into a gene. This particular DNA segment is now called insertion sequence 1, or IS1 , and the insertion
of IS1 into the genome is an example of a transposition event. E. coli contains a number of IS elements (e.g., IS1,
IS2, and IS10R), each present in up to 30 copies per genome and each with a characteristic length and unique
nucleotide sequence. IS1, for instance, is 768 bp long and is present in 4 to 19 copies on the E. coli chromosome.
Among bacteria as a whole, the IS elements range in size from 768 bp to more than 5,000 bp and are found in
most cells
 All IS elements end with perfect or nearly perfect terminal inverted repeats (IRs) of 9 to 41 bp. This means that
essentially the same sequence is found at each end of an IS, but in opposite orientations. The inverted repeats of
 IS1 are 23 bp long . When IS elements integrate at random points along the chromosome, they often cause
mutations by disrupting either the coding sequence of a gene or a gene’s regulatory region.
 Promoters within the IS elements themselves may also have effects by altering the expression of nearby genes.
In addition, the presence of an IS element in the chromosome can cause mutations such as deletions and
inversions in the adjacent DNA. Finally, deletion and insertion events can also occur as a result of crossing-over
between duplicated IS elements in the genome.
 The transposition of an IS element requires an enzyme encoded by the IS element called transposase. The
transposase recognizes the IR sequences of the element to initiate transposition.
 The frequency of transposition is characteristic of each IS element and ranges from to per generation.. Insertion
takes place at a target site with which the element has no sequence homology.
 First, a staggered cut is made in the target site and the IS element is then inserted, becoming joined to the
single-stranded ends. DNA polymerase and DNA ligase fill in the gaps, producing an integrated IS element with
two direct repeats of the target-site sequence flanking the IS element. In this case, direct means that the two
sequences are repeated in the same orientation. The direct repeats are called target-site duplications. Their size
is specific to the IS element, but they tend to be small (4 to 13 bp).

 Transposons. Like an IS element, a transposon (Tn) contains genes for the insertion of the DNA
segment into the chromosome and mobilization of the element to other locations on the chromosome.
 A transposon is more complex than an IS element in that it contains additional genes.
 There are two types of bacterial transposons: composite transposons and noncomposite transposons .
 Composite transposons, exemplified by Tn10( Tetracycline resistance), are complex transposons with a
central region containing genes (for example, genes that confer resistance to antibiotics), flanked on both
sides by IS elements (also called IS modules). Eg.( Tn9- Chloramphenicol resistance, Tn5- Kanamycin
resistance)
 Composite transposons may be thousands of base pairs long. The IS elements are both of the same type and
are called ISL (for “left”) and ISR (for “right”). Depending on the transposon, ISL and ISR may be in the same or
inverted orientation relative to each other. Because the ISs themselves have terminal inverted repeats, the
composite transposons also have terminal inverted repeats.
 Transposition of composite transposons occurs because one or both IS elements supply the transposase,
which recognizes the inverted repeats of the IS elements at the two ends of the transposon and initiates
transposition (as with the transposition of IS elements).
 Transposition of Tn10 is rare, occurring once in 107 cell generations. Like IS elements, composite transposons
produce target-site duplications after transposition. In the case of Tn10, the target-site duplications are 9bp
long.
 Noncomposite transposons , exemplified by Tn3, also contain genes such as those conferring resistance to
antibiotics, but they do not terminate with IS elements.
 However, at their ends they have inverted repeated sequences that are required for transposition. Enzymes
for transposition are encoded by genes in the central region of noncomposite transposons.
 Transposase catalyzes the insertion of a transposon into new sites, and resolvase is an enzyme involved in the
particular recombinational events associated with transposition.
 Like composite transposons, noncomposite transposons cause target-site duplications when they move. For
example, Tn3 produces a 5- bp target-site duplication when it inserts into the genome.
 First, the donor DNA containing the transposable element fuses with the recipient DNA to form a cointegrate.
Because of the way this occurs, the transposable element is duplicated and one copy is located at each
junction between donor and recipient DNA.
 Next, recombination between the duplicated transposable elements resolves the cointegrate into two
genomes, each with one copy of the element. Because the transposable element becomes duplicated, the
process is called replicative transposition (also called copyand-paste transposition).
 Tn3 and related noncomposite transposons move by replicative transposition
 . A second type of transposition mechanism involves the movement of a transposable element from one
location to another on the same or different DNA without replication of the element. This mechanism is
called conservative (nonreplicative) transposition (also called cutand-paste transposition).
 In other words, the element is lost from the original position when it transposes. Tn10 transposes by
conservative transposition. As with the movement of IS elements, the transposition of transposons can cause
mutations. The insertion of a transposon into the reading frame of a gene disrupts it, causing a loss-of-
function mutation of that gene. Insertion into a gene’s controlling region can cause changes in the level of
expression of the gene, depending on the promoter elements in the transposon and how they are oriented
with respect to the gene. Deletion and insertion events also result from the activities of the transposons and
from crossing-over between duplicated transposons in the genome
Mu transposon
 Temperate bacteriophage Mu(mutator) can cause mutations when it transposes, its structure includes
a. A 37 kb linear DNA in the phage particle that has central phage DNA and unequal lengths of host
DNA at the ends.
b. The DNA’s G segment can invert, and is found in both orientation in viral DNA. This segment
controls its infectivity and host range determining which host to infect in a given orientation.
 Following infection, Mu integrates into the host chromosome by conservative (non-replicative)
transposition.
 Integration produces prophage DNA flanked by 5bp target site direct repeats, flanking DNA from the
previous host is lost during integration.
 The Mu prophage now replicates when the E. coli chromosome replicates, due to phage encoded
repressor that prevents most Mu gene expression.
 Mu prophage stays integrated during the lytic cycle, and replication of Mu’s genome is by replicative
transposition.
 Mu causes insertion, deletion, inversions and translocations.
 Each Mu is packaged from a different site in the host genome, so the host DNA on the ends of Mu is
unique in every different phage head.
 Mu can also act as a genetic snap fastener, mobilizing any kind of DNA and transposing it anywhere in
the genome. For example – it can mobilize another phage(such as lambda) or the Ffactor, in such
situations, the inserted DNA is flanked by two Mu genomes.