Transcript Sequence Alignment - Faculty of Science at Bilkent University

Transposable Elements
IS
P-elements
Human repetitive sequences
Prokaryotes
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Insertion Sequences (IS elements)
Composite transposons
Tn3 elements
IS element
IS elements
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IS elements are relatively small
transposable elements that range in size
from 760 to less than 2,500 base pairs (bp).
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can insert at many different sites in bacterial and
viral chromosomes and plasmids, and they
contain genes whose products are involved in
promoting and regulating transposition.
One of the genes is a transposase that
functions in excision of the element from a
chromosome, plasmid.
IS elements
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IS elements typically generate
unstable mutants that revert to wildtype at a detectable frequency. For
that reason, IS elements originally
were called "mutable" genes.
IS elements
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All IS elements contain inverted
terminal repeats that range in size
(length) from 9 to 40 base pairs.
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At the site of integration there invariably
is a target site duplication of from 2-13
base pairs.
Composite Transposons
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Composite transposons (denoted by symbol
Tn):
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Tn elements stem from two IS elements that
insert near one other. The regions (sequences)
between the two elements can be "mobilized" by
the joint action of the two IS elements. This is of
significance in that many Tn elements possess
genes that confer resistance to antibiotics
between the two IS elements.
Tn transposition is regulated by a
"repressor" that appears to exist to keep the
elements somewhat quiescent.
Tn3
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Tn3 elements are simply large
transposable elements that are not
generated by flanking IS elements (as
in Tn elements).
Eukaryotes
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DNA transposable elements
RNA transposable elements
Conservative Transposition
http://nitro.biosci.arizona.edu/courses/EEB600A-2003/lectures/lecture26/lecture26.html
Replicative Transposition
Retrotransposition
AC/DS elements in maize
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AC is a full-length autonomous copy
DS is a truncated copy of AC that is
non-autonomous, requiring AC in order
to transpose
Transposable Elements
(Transposons)
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DNA elements capable of moving ("transposing")
about the genome
Discovered by Barbara McClintock, largely from
cytogenetic studies in maize, but since found
in most organisms
She was studying "variegation" or sectoring in
leaves and seeds
She liked to call them "controlling elements“
because they affected gene expression in
myriad ways
Mutant Kernel Phenotypes
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Pigmentation mutants
 affect anthocyanin pathway
 elements jump in/out of transcription
factor genes (C or R)
 sectoring phenotype - somatic mutations
 whole kernel effected - germ line
mutation
Some maize phenotypes caused by
transposable elements excising in somatic
tissues.
Start with mutant kernels defective in anthocyanin synthesis and
the element excises during development.
Somatic Excision of Ds from C
Wild type
Mutant
Sectoring
Molecular Analysis of Transposons

Transposons isolated by first cloning a gene that
they invaded. A number have been cloned this way,
via "Transposon trapping“.
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Some common molecular features:
 Exist as multiple copies in the genome
 Insertion site of element does not have extensive
homology to the transposon
 Termini are an inverted repeat
 Encode “transposases” that promote movement
 A short, direct repeat of genomic DNA often
flanks the transposon : “Footprint”
Ac and Ds
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Ds is derived from Ac by internal deletions
Ds is not autonomous, requires Ac to move
Element termini are an imperfect IR
Ac encodes a protein that promotes
movement - Transposase
Transposase excises element at IR, and also
cuts the target
Structure of Ac and Ds deletion
derivatives
Ds is not autonomous, requires Ac to move! Fig. 23.1
How duplications
in the target site
probably occur.
Duplication
remains when
element excises,
thus the
Footprint.
Hybrid Dysgenesis
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P-elements are transposable
elements that carry genes for
transposase activity that
cause the elements to move,
and repressor activity that
prevents expression of
transposase.
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In a cross between a Pelement-carrying female and
a laboratory male [left],
repressors in the maternally derived cytoplasm repress
expression of the maternally
- inherited P elements. The
resulting offspring show the
wild-type phenotype.
Examples: P-elements in
Drosophila
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P elements were discovered when it was found that
certain strains of Drosophila exhibited an
assortment of aberrant phenotypes, including
elevated mutation (and reversion), chromosome
breakage, and sterility = hybrid dysgenesis
normally (within populations) the P elements are
quiescent and do not “jump.” When “hybrids” were
made between individuals from different geographic
populations, the elements “moved” and promoted
the dysgenic phenotypes.
P-elements
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P elements vary in size (the largest are
nearly 3,000 base pairs in length).
Complete (intact) P elements possess
a gene for a transposase. The number
of P elements per individual varies
from a few to up to 50.
Experimental uses of Pelements
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Transposon tagging, where genes mutated by P
element insertion can be isolated and "discovered"
by using the P element sequence as a "tag”; and
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Transformation vectoring, where genes or
sequences of interest are "vectored" into a
chromosomal location by putting the
gene/sequence of interest into an incomplete P
element (no transposase) and carrying out a mixed
infection (transformation or electroporation) with a
complete P element.
Mariner elements
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Mariner elements appear to be a fairly
widespread transposon of roughly
1,200 base pairs.
Retrotransposones
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Retrovirus-like elements
Retroposons
LTR=long terminal repeat
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Flanks three genes; a complete retrovirus
has three genes
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gag = structural gene for capsid
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Pol = reverse transcriptase plus other stuff.
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env = envelope gene for the virus
retrovirus
Retrovirus like elements
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The basic structure of retrovirus-like
elements is a central coding region of two
genes flanked by long terminal repeats
[LTRs] that are oriented in the same
direction and bounded by short inverted
repeats.
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The two genes are homologous to two
genes in retroviruses and encode a
structural protein of the virus capsule and a
reverse transcriptase/integrase enzyme.
Retrovirus like elements
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Active retroviruses carry a third gene that
codes for a protein of the virus envelope.
Active retroviruses are capable of exiting
cells and infecting other cells.
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Transposition involves transcription (RNA
synthesis) of the DNA sequence integrated
in the chromosome, reverse transcription of
the RNA, synthesis of a double-stranded
DNA from the RNA, and insertion into a new
chromosomal location.
Retroposons
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These are elements that move through an RNA
intermediary but do not possess direct or inverted
repeats at their termini (LTR) nor the env gene.
They possess instead a string of A=T base pairs at
one end (of the DNA), and presumably represent a
copy from reverse transcription of the poly-A tail of
the mature RNA transcript.
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Some LINE sequences in mammals are retroposons, and
the LlNE-1 retroposon is the only transposable element
thus far documented in humans.
Drosophila telomere sequences
Non-LTR
transposition
The LINE is transcribed into mRNA (red). A
part of this mRNA is translated into proteins
involved in the integration complex, which
binds to the 3' end of the mRNA transcript.
The target site (blue) is cleaved followed by
reverse transcription, with the 3' end of the
target site as the primer. Newly synthesized
cDNA is shown in pale green. Ligation of the
cDNA occurs at the 5' end, and the second
strand is synthesized using the first cDNA
strand as template and the host DNA
polymerase
Long term fate of non-LTR
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In an evolving genome, non-LTR elements are
thought to proliferate by amplification of an
extremely small number of "master" genes.
These genes usually give rise to inactive copies
(truncated at the 5' end) that are incapable of
further transposition within the genome.
The defective copies arise because of their mode of
transposition through reverse transcription (see the
figure), which in most cases stops replication before
the 5' end is reached.
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These truncated elements, called DOA ("dead on arrival"),
can be used as surrogates for pseudogenes in species
such as Drosophila that have few bona fide pseudogenes
LINEs
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In humans, are about 6 kb long, harbour an internal
polymerase II promoter and encode two open
reading frames (ORFs).
Upon translation, a LINE RNA assembles with its
own encoded proteins and moves to the nucleus,
where an endonuclease activity makes a singlestranded nick and the reverse transcriptase uses
the nicked DNA to prime reverse transcription from
the 3' end of the LINE RNA.
Reverse transcription frequently fails to proceed to
the 5' end, resulting in many truncated,
nonfunctional insertions.
LINEs
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Most LINE-derived repeats are short, with an average size of
900 bp for all LINE1 copies, and a median size of 1,070 bp for
copies of the currently active LINE1 element (L1Hs).
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The LINE machinery is believed to be responsible for most
reverse transcription in the genome, including the
retrotransposition of the non-autonomous SINEs and the
creation of processed pseudogenes
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Three distantly related LINE families are found in the human
genome: LINE1, LINE2 and LINE3.
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Only LINE1 is still active.
SINEs
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SINEs are freeloaders on the backs of LINE
elements.
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short (about 100-400 bp), harbour an
internal polymerase III promoter and encode
no proteins.
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non-autonomous transposons use the LINE
machinery for transposition.
SINEs
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most SINEs 'live' by sharing the 3' end
with a resident LINE element.
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Most promoter regions of known
SINEs are derived from tRNA
sequences
Alu
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A single monophyletic family of SINEs (ALU)
derived from the signal recognition particle
component 7SL
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This family is the only active SINE in the
human genome
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The human genome contains three distinct
monophyletic families of SINEs: the active
Alu, and the inactive MIR and Ther2/MIR3.
Human Genome
Comparative
Element
Human
Fly
Worm
Arabodopis
LINE/SINE
33.4%
0.7%
0.4%
0.5%
LTR
8.1%
1.5%
0.0%
4.8%
DNA
2.8%
0.7%
5.3%
5.1%
All TEs
44.4%
3.1%
6.5%
10.5%
Direct Repeats

duplication of a
short sequence at
the target site. This
generates short
direct repeats
flanking the newly
inserted element.
This results for a
staggered cut
being made in the
DNA strands at the
site of insertion
LTR retroposons
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Bascially, these are retroviruses
without the env protein.
Current thinking is that retroviruses
evolved from retroposons.
They have the LTR and (usually) gag
genes. LTR retroposons are often
simple called retrotransposons.
Simple Sequence Repeat
Content of Human Genome
Human Genome
Mouse vs Human Genome
Transposable Elements
Transposable Elements
Activity of Transposable
Elements
Evolution of TEs
LTRs
Transposons
LINEs
LINEs
SINEs
LINE/SINE Genomic
Distribution
LINE distribution
SINE distribution
CpG Methylation
CpG Methylation
CpG Islands
CpG Islands
Function
Methylation and Gene
Silencing
Methylation and CpG Content
Diseases
Spontaneous Mutations
CpG Mutations
CpG Mutations
CpG Mutations
Function of Alu
Function of Alu