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The traditional view of the DNA of a cell has been that of a fixed sequence of bases that is only subject to occasional
changes by means of mutation or by recombination when cells exchange genetic material. There has therefore been a
tendency to think of all cells in a pure bacterial culture as having an identical genetic make-up. It is now known that
this is far from the truth. The genetic material is much more fluid than that and is subject to a range of larger-scale
alterations in its structure, including insertions, transpositions, inversions and deletions. Some of these variations
are readily reversible and generate high levels of genetic diversity
The world of transposable elements is rich with diversity. Many different types of elements have been identified in
assortment of organisms. They form prominent parts of the genome and clearly have roles in modifying the structure of
chromosomes and in modulating the expressions of genes
Transposons are segments of DNA that can move around to different positions in the genome of a single cell. In the
process, they may cause mutations, increase (or decrease) the amount of DNA in the genome.
These mobile segments of DNA are sometimes called "jumping genes".
Transposons cannot exist independent of a replicon (because they don't have an ori of their own)
Transposition
Transposase must be able to:
1) Recognize the inverted repeats and
2) Recognize the target sequence (or a partial match of the target sequence)
Target sequence is 5-9 bp on the recipient DNA molecule
Conservative transposition
Requires Transposase
1) Transposase cuts transposon out of donor DNA (blunt cut)
2) Transposase makes a staggered cut at the target sequence
3) Transposase ligates transposon into the target
4) Gaps are filled in with DNA pol I and ligase
Results:
Target DNA has been duplicated in process
Usually destroys donor DNA
Replicative transposition
Requires transposase, resolvase and an IRS (internal resolution site) recognized by resolvase
1) Transposase makes nicks at ends of transposon and at ends of target
2) Transposase ligates the donor and target together such that they're linked together via single strands of the transposon
3) Gaps are filled in with host DNA pol I and ligase (transposon is replicated)
4) Resolvase catalyzes recombination between the 2 IRSs
Results:
Transposon does not leave the donor DNA so donor is not destroyed
Transposon is replicated so 2 copies of the
transposon are produced
Depending upon how they transpose; There are
three distinct types:
Class I Transposons consisting only of DNA that
moves directly from place to place.
Class II Transposons; also known as Miniature
Inverted-repeats Transposable Elements or MITEs.
Class III Retrotransposons that first transcribe the
DNA into RNA and then use reverse transcriptase to
make a DNA copy of the RNA to insert in a new
location.
Class I Transposons
They usually move by a mechanism analogous to cut and paste where the element is physically cut out to transpose it to
a new position. The new position is situated any where in the cells genome using the transposase enzyme which is
encoded by the element itself. This mechanism also called cut and paste mechanism. Different types of Transposase
work in different ways. Some can bind to any part of the DNA molecule, and the target site can therefore be anywhere,
while others bind to specific sequences
Transposase binds to:
1) Both ends of the transposon, which consist of inverted repeats; that is, identical sequences reading in opposite
directions.
2) A sequence of DNA that makes up the target site. Some transposases require a specific sequence as their target site;
other can insert the transposon anywhere in the genome.
Transposase makes a staggered cut at the target site producing sticky ends produced by some restriction enzymes cuts
out the transposon and ligates it into the target site.The gaps are filled in by Watson-Crick base pairing by DNA
polymerase and DNA ligase closes the sugar-phosphate backbone. This results in target site duplication and the
insertion sites of DNA transposons may be identified by short direct repeats (a staggered cut in the target DNA filled by
DNA polymerase) followed by inverted repeats (which are important for the transposon excision by transposase).
Often transposons lose their gene for transposase; but as long as somewhere in the cell there is a transposon that can
synthesize the enzyme, their inverted repeats are recognized and they, too, can be moved to a new location. These
transposons undergo conservative transposition
The transposons which only move by cut and paste may duplicate themselves if the transposition happens during S
phase of the cell cycle when the "donor" site has already been replicated, but the "target" site has not.
Class II Transposons
Replication of transposable elements is involved in these elements transposition by Transposase which interacts
between the element and the potential insertion site. During the interaction the element is replicated and one copy of it
is inserted in the new site. The other copy remains in the original site. This mechanism is also called replicative
transposition
Transposase makes a staggered cut at the target site producing sticky ends produced by some restriction enzymes
ligates it into the target site.The gaps are filled in by Watson-Crick base pairing by DNA polymerase and DNA ligase
closes the sugar-phosphate backbone. This results in target site duplication and the insertion sites of DNA transposons
may be identified by short direct repeats (a staggered cut in the target DNA filled by DNA polymerase) followed by
inverted repeats (which are important for the transposon excision by transposase). These transposons undergo
replicative transposition
Composite transposons
They are denoted by the symbol Tn and are created when 2 IS sequences are inserted beside each other. The region
between them can be transported along with the inserted IS. Hence the ISs have made an immobile gene mobile. These
genes are nothing related to transposition they mostly code for resistance to a certain antibiotic
These composite transposons consist of two copies of an insertion sequence on either
side of a set of resistance genes. For example the tetracycline resistance transposonTn10,
which is about 9300 bp in length, consists of a central region carrying the resistance
determinants flanked by two copies of the IS10 insertion sequence in opposite orientations
IS10 itself is about 1300 bp long with 23-bp inverted repeat ends and contains a Transposase
gene.
Composite
transposons may
have their flanking
IS regions in
inverted orientation
or as direct repeats.
For example Tn10
and Tn5 both have
inverted repeats of
an IS (IS10 and IS50
respectively) at their
ends, while Tn9 has
direct repeats of IS1.
The transposition behaviour of such composite elements can be quite complex; the insertion
sequences themselves may transpose independently or transposition of the entire region may
occur. Furthermore, recombination between the IS elements can occur, leading to deletion or
inversion of the region separating them .Even more complex arrangements can occur. For
example, Tn4 appears to be related to Tn21 but contains a complete copy of Tn3 within it. The
ampicillin resistance gene of Tn4 can thus be transposed as part of the complete Tn4
transposon or by transposition of the Tn3 element. Thus several layers of transposons can
occur, nested within one another.
In Tn5 the IS50R is capable of producing Transposase but IS50Lcannot due to deletion of a
nucleotide pair.Tn5 when carried by non lytic bacteriophage the frequency of transposition is
reduced if the virus already carries a copy on Tn5.Sinc the resident Tn5 inhibits the entry of
incoming Tn5 by synthesis of a repressor
Tn 3 elements
These are larger than IS elements but are different from composite transposons due to
absence of IS element flanks. instead have terminal inverted repeats. The structure of Tn3; it
consists of about 4957 base pairs and has a short (38 bp) inverted repeat sequence at each
end. It is therefore
analogous to an insertion
sequence, the distinction
being that a transposon
carries an identifiable
genetic marker – in this
case the ampicillin
resistance gene (bla, β-lactamase). Tn3 codes for two other proteins as well: a Transposase
(TnpA), and TnpR , resolvase a bi functional protein that acts as a repressor and is also
responsible for one stage of transposition known as resolution. There is a short direct repeat at
either end of the transposon (five base pairs in the case of Tn3).
Transposition of Tn3 occurs in 2 stages. Firstly the transposase fuses 2 circular genomes one with Tn3 and the other
without it. Resulting structure is called Cointegrate during this process Tn3 replicates hence one copy of Tn3 is
rendered to each genome which are oriented in the same direction. Secondly Resolvase bring about recombination
between the 2 Tn3 elements in the Cointegrate at the res (resolution site) resulting in two circular genomes each with a
copy of Tn3.Resolvase also prevents the expression on the two genes by binding at the res site interfering with the
transcription of the genes creating a chronic shortage of the products hence Tn3 remains to be Immobile. These also
come under the category replicative transposons
Transposons in Eukaryotes
There are many different types of transposons in Eukaryotes. Some are abundant in the genome others are rare. These
elements have inverted repeats and direct repeats and also encode transposase for the mobility of the transposase
Female gametes:
__________________________________
C bz
Male gametes:
__________________________________
CI Bz ds
The following is the expected chromosomal composition of triploid endosperm. Because of the dominant CI allele the
endosperm should be colorless without breakage
____________________________ (from female)
C bz
____________________________ (from female)
C bz
___________________________ (from male)
CI Bz ds
But if breakage at ds occurred, then the genotype of the endosperm would be:
____________________________ (from female)
C bz
____________________________ (from female)
C bz
….……………………… _______ (from male)
and any cells with this genotype would be dark brown in color.
Breakage at Ds had been established by McClintock prior to performing these experiments. The designation Ds
was short for dissociation or a locus were breakage of chromosomes occurred. But after crossing with a number of
different genetic stocks, she realized that Ds alone could not induce the breakage. A second factor, Ac, short for
activator, was also necessary since its transposase causes the breakage of the chromosome. Ds can be activate dif Ac
transposase is present anywhere in the genome (Thus, some genetic stocks contained Ac whereas other stocks did not
contain that locus.) This system is called a two-element system and historically has been called the Ac/Ds system.
Additional genetic stocks were analyzed by McClintock and she determined that in the presence of Ac, Ds could
move locations as well as cause breakages. She was able to isolate a corn line where Ds had moved into the normal Bz
allele and caused a mutation in that gene. But as was mentioned this only occurred when Ac was present. Furthermore,
when this new line was used and Ac was present, the Ds element was shown to move out of the Bz locus and reversion
to the original phenotype was detected. This mutated allele was designated bzm1. But in the absence of Ac, bzm1 was a
stable allele. Another unstable Bz allele was found that contained an Ac insertion and was designated bzm2. One
difference between this allele and bzm1 was its higher rate of transposition and reversion back to the original phenotype
.Ac/Ds are also called controlling elements since there insertion into various position renders the nearby gene altered or
inactive. Multiple copies of these are present in the genome
. So the conclusions drawn from these experiments and observations are:
1) Ds requires some factor provided by Ac to move, whereas Ac is independent
2) Because of their relationship, Ac is termed an autonomous element and Ds a non- autonomous element.
3) Because both Ac and Ds can move, they are called transposable genetic elements.
About 50% of the total genome of maize consists of transposons. The Ac/Ds system McClintock described are
class II transposons.
Ac is 4563 bp in length contain essential 11-bp inverted repeats at the ends encodes a 3.5 kb mRNA that is
translated into a 92 kDa transposase protein 101 N-terminal amino acids not required for transposition about 200 bp on
each end of element necessary for transposition transposase binds to the hexamer AAACGG 8-bp direct repeats of
target DNA are generated footprints (residual DNA sequences) are often left behind after the element is excised Ds are
truncated versions of Ac; these can lack a transposase or the inverted repeat. Ds is structurally heterogeneous and have
similar IRs but the internal sequence varies. Some Ds are derived from Ac by loss of certain internal sequences. Other
Ds contain non-Ac DNA between their IRs. Other Ds types are Aberrant Ds, piggyback arranged Ds where one Ds
element is inserted into an other but in the inverted orientation hence also called double Ds elements
Drosophila – P elements
A P element is a transposon that is present in the fruit fly Drosophila melanogaster and is used widely for mutagenesis
and the creation of genetically modified flies used for genetic research. The P element gives rise to a phenotype known
as hybrid dysgenesis.
P elements seem to have first appeared in the species only in the middle of the twentieth century. Over the last fifty
years, they have spread through every wild population, so that only older laboratory stocks lack them. It was identified
by geneticists Margaret and James Kidwell in 1977
Hybrid dysgenic mutant’s white gene was compared with that of recessive white gene and found that the former had a
small element inserted. These were the P elements
Characteristics
The P element is a class II transposon, which means that its movement within the genome is made possible by a
transposase. The complete element is 2907 bp and is autonomous because it encodes a functional transposase; non-
autonomous P elements which lack a functional transposase gene due to mutation also exist. Non-autonomous P
elements can still move within the genome if there are autonomous elements to produce transposase. The P element can
be identified by its terminal 31-bp inverted repeats, 11-bp subterminal inverted repeat and the 8 bp direct repeats its
movement into and out of DNA sequence produces.
Several lines of evidence show that P elements transpose non replicatively and without an RNA intermediate donor
element is excised and reinserted into a recipient site creating a direct duplication of 8 bp at the site of insertion
P element insertions have been found at thousands of genomic positions, but not all sites are equally likely to be hit.
The mechanism of insertion site selection is not known, but several generalizations can be made:
(i) Euchromatic sites are hit more often than the heterochromatin
(ii) Some euchromatic loci are much more susceptible to P mutagenesis than others. For example, the singed gene is hit
at frequencies approaching 10-2 whereas the vestigial gene has a rate of less than 10-6 .Despite this variability, there is no
evidence that any loci are immune from P element mutagenesis given a sufficiently large sample size;
(iii) Within genes there is a preference for insertion in the non coding upstream sequences
(iv) Target sites with close matches to the consensus octamer GGCCAGAC are more likely to receive P element
insertions
(v) P elements tend to insert into or near other P elements, with a particular preference for base pairs 19-26 of the
target P element
(vi) Some P elements have been observed to jump preferentially to sites closely linked to the donor site
The P element transposase is an 87 kiloDalton protein encoded by autonomous P elements .It binds to subterminal
regions at both ends of the element and represses transcription. GTP is also bound by the transposase, and is required
for transposition in vitro .
Some natural populations contain a few P elements, but others contain up to 50 copies. Interestingly, strains captured
before 1950 do not contain any P elements. These are called "empty" strains. These strains may represent the primitive
condition and P elements may have invaded these natural populations. Close relatives of the lab species D.
melanaogaster have maintained the empty condition, but more distantly-related strains like D.willostoni acquired the
element. It has been suggested that the element entered the species by piggy-backing on viruses that naturally infect
Drosophila
Hybrid dysgenesis
When P elements are mobilized they produce a syndrome of traits known collectively as hybrid dysgenesis
Hybrid dysgenesis refers to the high rate of mutation in germ line cells of Drosophila strains resulting from a cross of
males with autonomous P elements (P Strain/P cytotype) and females that lack P elements (M Strain/M cytotype). The
hybrid dysgenesis syndrome is marked by temperature-dependent sterility, elevated mutation rates, and increased
chromosome rearrangement and recombination.
The hybrid dysgenesis phenotype is affected by the transposition of P elements within the germ-line cells of offspring
of P strain males with M strain females. These two kinds of strains are called "P" and "M" because they contribute
paternally and maternally respectively to hybrid dysgenesis. Transposition only occurs in germ-line cells, because a
splicing event needed to make transposase mRNA does not occur in somatic cells.
Hybrid dysgenesis manifests when crossing P strain males with M strain females and not when crossing P strain
females (females with autonomous P elements) with M strain males. The eggs of P strain females contain high amounts
of a repressor protein that prevents transcription of the transposase gene. The eggs of M strain mothers, which do not
contain the repressor protein, allow for transposition of P elements from the sperm of fathers.
The dysgenic traits can be explained largely by genomic changes due to P element transposition and excision in
developing germ cells. The sterility is due to loss of germ cells early in development It is more pronounced in females,
where there are fewer germ cells to spare than in males, and at temperatures above 25°C. The mutations come about
through several mechanisms, but are primarily P insertions into genes and imprecise excision of P elements near genes
Chromosome rearrangements usually result from breakage at the sites of two or more P element insertions, followed
by rejoining of the chromosome segments in a different order). P-induced recombination occurs preferentially in the
genetic intervals containing mobile P elements, and usually within 2 kb of the insertion site
P element movement within the fly is a maternally inherited trait. Movement is suppressed in flies with the P cytotype,
but is permissible in the M cytotype. Furthermore, P-cytotype flies contain P elements, whereas M-cytotype elements
do not. When movement does occur it happens in the germ line, that is those cells which produce the gametes. This
movement of P elements creates many different changes in phenotype, but few are fatal. The progeny are described as
being dysgenic that refers to biological deficiencies that are a result of the P element movement. Movement is
suppressed in somatic cells where the effects would be more damaging this is because at the level of RNA splicing
metabolic block occurs hence one of the introns remains in the transcript and is prevented from translation of the
transposase gene by a stop codon that prematurely terminates translation. This condition of germ line abnormalities
which generates mutations, chromosomal breakage and sterility is called P-M hybrid dysgenesis and is mediated by P
element movement.
P elements are repressed not only within P strains, but also in the hybrids of P(female) x M(male) crosses. They are not
repressed in hybrids from the reciprocal cross, suggesting that repressor products produced in the P strain germline can
be inherited maternally. Interestingly, this effect goes beyond simple maternal inheritance. Progeny from the cross
MP(female) x P(male) show more P mobility than those from the cross PM(female) x PM(male) where "MP" and "PM"
represent the two reciprocal F1 hybrids with the female component shown first. Thus, the repressed state, called the P
cytotype, is jointly determined by chromosomal and maternal components. One explanation proposed for this unusual
inheritance was that the repressor-making P elements in the MP hybrids are more likely to be excised than those of the
PM hybrids However, that explanation was ruled out by the finding that a single repressor-producing P element was
sufficient for this mode of inheritance An alternative model involving differential splicing of the same intron that is
involved in tissue-specific regulation, provides an adequate explanation for available data..
Yeast – Ty elements
This element has several features that are unique, and it appears to resemble a primitive retrovirus. A retrovirus is a
RNA virus that, after being uncoated in the host cell, converts its RNA to a DNA copy by the enzyme reverse
transcriptase. This enzyme is encoded by the retroviral pol gene. The DNA copy of the retrovirus is inserted into the
eukaryotic genome, and it remains there as a provirus until it is excised and undergoes transcription to produce new
viral particles.
Features of TY elements
• about 35 copies in the haploid yeast gene
• They about 5900 bp long and are bounded at each end by δ sequence
• δ sequence has 340 bp sequence and has orientation in the same direction; these are called long terminal repeats
or LTRs
• They are flanked by 5 bp direct repeats
• Target sit duplication are rich in A:T bp
• They resemble eukaryotic retroviruses; because they lack some of the retroviral functions they are considered to be
primitive retroviruses; because of their similarities they are called retrotransposons
• transposition involves an RNA intermediate that is generated by transcription of the TY element; a reverse
transcriptase (encoded by the TyB gene of the element) makes a DNA copy of the element which is then inserted
into a new site in the yeast genome
Retrotransposons
Retroviruses (HIV-1)
HIV-1 — the cause of AIDS — and other human retroviruses (e.g., HTLV-1, the human T-cell leukemia virus) behave
like retrotransposons.
The RNA genome of HIV-1 contains a gene for reverse transcriptase and one for integrase.
The integrase serves the same function as the transposases of DNA transposons. The DNA copies can be inserted
anywhere in the genome.
Molecules of both enzymes are incorporated in the virus particle.
Retroviruses like elements
They are found in many different organisms including yeast plants and animals. Despite difference in size and
nucleotide sequence, they all have same basic structure. A central coding region flanked by long terminal repeats (LTR)
which are oriented in the same direction. Each LTR is bounded by short inverted repeats. The coding region contains a
small number of genes usually two. One is homologous to gag (encodes structural protein of virus capsule) the other is
homologous to pol(encodes reverse transcriptase/ integrase protein).Retrovirus have a third gene env (encodes a protein
component of the virus envelope)
Retroposons
These are also called non LTR transposons. They are large widely distributed transposons. They move through an RNA
molecule and then reverse transcribed into DNA. They create target site duplication but do not have inverted or direct
repeats as integral parts of their termini. They are distinguished by a homogenous sequence of A:T base pairs ant one
end. This sequence is derived from the poly(A) tail that is added near the 3’ of the retroposon RNA during its
maturation. Integrated retroposons therefore exihibit a vestige of their origin as reverse transcripts of polyadenylated
RNAs
1.LINEs (Long interspersed elements)
The human genome contains some 850,000 LINEs (representing some 21% of the genome).
Most of these belong to a family called LINE-1 (L1).
These L1 elements are DNA sequences that range in length from a few hundred to as many as 9,000 base pairs.
Only about 50 L1 elements are functional "genes"; that is, can be transcribed and translated.
The functional L1 elements are about 6,500 bp in length and encode three proteins, including an endonuclease that cuts
DNA and a reverse transcriptase that makes a DNA copy of an RNA transcript.
L1 activity proceeds as follows:
RNA polymerase II transcribes the L1 DNA into RNA.
The RNA is translated by ribosomes in the cytoplasm into the proteins.
The proteins and RNA join together and reenter the nucleus.
The endonuclease cuts a strand of "target" DNA, often in the intron of a gene.
The reverse transcriptase copies the L1 RNA into L1 DNA which is inserted into the target DNA forming a new L1
element there.
Through this copy-paste mechanism, the number of LINEs can increase in the genome.
The diversity of LINEs between individual human genomes makes them useful markers for DNA "fingerprinting".
Variation in the length of L1 elements:
Transcription of active L1 elements sometimes continues downstream into additional DNA producing a longer
transposed element.
Reverse transcription of L1 RNA often concludes prematurely and produces a shortened transposed element.
While these elements are not functional, they may play a role in regulating the efficiency of transcription of the gene in
which they reside .
Occasionally, L1 activity makes and inserts a copy of a cellular mRNA (thus a natural cDNA). Lacking introns as well
as the necessary control elements like promoters, these genes are not expressed. They represent one category of
pseudogene.
2.SINEs (Short interspersed elements)
SINEs are short DNA sequences (100–400 base pairs) that represent reverse-transcribed RNA molecules originally
transcribed by RNA polymerase III; that is, molecules of tRNA, 5S rRNA, and some other small nuclear RNAs.
The most abundant SINEs are the Alu elements. There are over one million copies in the human genome (representing
about 11% of the total DNA).
Alu elements consist of a sequence of 300 base pairs containing a site that is recognized by the restriction enzyme AluI.
They appear to be reverse transcripts of 7S RNA, part of the signal recognition particle.
Most SINEs do not encode any functional molecules and depend on the machinery of active L1 elements to be
transposed; that is, copied and pasted in new locations.