L23_Transposons15 (1)
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203.341 Transposons and Genetic Conflict Lectures 23 and 24 Prak and Kazazian, 2000. Nature Reviews Genetics 1:134- Cordaux & Batzer, 2009 Nature Reviews Genetics 10:691 Austen Ganley, October 12 th , 2015
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Transcript of L23_Transposons15 (1)
203.341!Transposons and Genetic
Conflict!Lectures 23 and 24!
!
Prak and Kazazian, 2000. Nature Reviews Genetics 1:134-!
Cordaux & Batzer, 2009 Nature Reviews Genetics 10:691!
Austen Ganley, October 12th, 2015
Barbara McClintock • Barbara McClintock is famous for
discovering transposons in maize!• However, she was a brilliant
cytogeneticist, who made many other important discoveries!
Controlling elements/transposons • McClintock’s discovery of transposons showed that the
genome was not static, but dynamic!• She thought these “jumping genes” controlled the expression
of many genes in the genome, and called them “controlling elements”!
• This was the first suggestion that suites of genes can be regulated to control processes such as development!
• She was also probably the first one to understand the idea of epigenetics (she called this “phase changes”)!
• Many of her ideas were ignored, but with the advent of molecular biology, people recognised their importance and she won the Nobel Prize in 1983!
www.bbc.co.uk/scotland/education/bitesize/higher/biology/control_regulation/genetic_control1_rev.shtml
Ac/Ds elements in maize • The break is
caused by a transposon at that site!
• Requires an element called Ac (activator)!
• Sometimes, yellow corn with purple spots were found!
• Led to evidence for a “jumping gene”!
Ds/Ac mechanism • The original line was produced by the Ds element jumping (transposing) into the “C” gene!
• The gene can continue transposing with the help of the Ac element, thus some patches of tissue will have the purple colour reappear!
• McClintock showed that the Ac element itself can transpose, too!
Feschotte et al., 2002. Nature Reviews Genetics 3: 329-
Weaver, Molecular Biology, 2008
Transposons are found throughout life • Transposons are found
throughout life!• In bacteria, often called
“IS” (insertion sequences)!• Many are related to viruses:
move between genomes, using viruses as a vector!
• Many different kinds of transposons; divided into families, with each family thought to have a common origin!
gydb.uv.es/phylogeny.php?tree=gagpol
Transposon types • Three classes of transposon -
class I (retrotransposons), class II (DNA transposons and MITEs), and helitrons!
• Class I and II transposons can both be further divided into two types: autonomous and non-autonomous!
• Autonomous transposons encode proteins that catalyse their transposition!
• Non-autonomous transposons do not, and rely on proteins from autonomous transposons of the same family for their transposition!Slotkin & Martienssen, 2007. Nature
Reviews Genetics 8: 272-
• Usually more non-autonomous versions of transposons than autonomous within a genome!
• Only a few autonomous transposons can catalyse the transposition of many non-autonomous transposons!
Feschotte et al., 2002. Nature Reviews Genetics 3: 329-
Autonomous vs Non-autonomous
Class II transposons • All DNA-based
transposons have small (10-200bp) inverted repeats at their ends. There are two types:!
• Conservative transposons move by cutting themselves out of the DNA, and re-inserting at a new site (cut-and-paste)!
• Replicative transposons move by making a copy of themselves, and integrating the copy into a new site (copy-and-paste)!
• Which is more mutagenic?!www.math.princeton.edu/~jgevertz/public_html_Pelement/three_transposition_methods.htm
Conservative transposition • You should recognise that
the Ds/Ac system in maize must work through conservative transposition!
• In the Ds/Ac system, the Ac transposase binds to the inverted repeats of the transposon, !
• It cuts the transposon out (hence sometimes resulting in broken chromosomes)!
• It then catalyses its insertion into a new, essentially random, integration site!
Replicative transposition • The replicative transposition
mechanism is a bit more complex!• Transposase makes cuts in the DNA
containing the transposon and the target site, joining these together, and then replicating the transposon DNA!
• Results in the transposon remaining in its original site, and a copy being made to the target site!
• Mechanism is related to homologous recombination; DNA synthesis produces two copies of the transposon from one!
Target site duplications • When transposons move,
they integrate into a new site by making staggered cuts at the integration site!
• Transposon then inserts, and the single-strand gaps are filled!
• Results in duplications of the target site!
• Duplications are typically small (3-30 bp), but are diagnostic of transposons!
www.sbs.utexas.edu/herrin/bio344/
Class I transposons - retroposons • Retrotransposons
transpose through an RNA intermediate!
• Autonomous retrotransposons contain a reverse transcriptase gene!
• Retrotransposon is transcribed, reverse transcribed, and the DNA copy integrates elsewhere in the genome!
• What type of transposon must all class I transposons be?! www.sbs.utexas.edu/herrin/bio344/
Evidence for retrotransposons
• Retrotransposition is fairly-easily demonstrated experimentally!
• An intron can be inserted into the retrotransposon, e.g. into a Ty element in yeast!
• When this element transposes, the new copy no longer has the intron!
Effects on the genome - summary
Prak & Kazazian, Nature Reviews Genetics, 2000, 1: 134-
• Insertion mutation!
• Target site mutation!
• Movement of genes!
• Recombination duplicates or deletes genes; genomic instability!
• Deletion or duplication of genes between transposons!
• The cell tries to control transposition, e.g. siRNA!
Effects on the genome - summary
Retroposons in the human genome
• Retrotransposons make up the vast majority of repetitive elements in the human genome!
• Also make up almost half of the human genome!
International Human Genome Sequencing Consortium, Nature 409: 860-921 (2001)
LINEs/SINEs/LTRs
• Three main types of retrotransposons:!• LINEs (long interspersed nuclear elements)!• SINEs (short interspersed nuclear elements)!• LTRs (long terminal repeat retrotransposons)!
Han & Boeke, 2005. BioEssays 27: 775-
LINEs
• LINEs have a 5’ UTR that acts as a promoter!• Encode two proteins: a DNA-binding protein, and a
reverse transcriptase with endonuclease activity!• Have a 3’ UTR region, followed by a poly-A tail!• Create 7-20 bp target site duplications!• Both autonomous and non-autonomous LINEs exist!
biol.lf1.cuni.cz/ucebnice/en/repetitive_dna.htm
• LINEs are transcribed into RNA, and then move into the cytoplasm and are processed, and bind to the two transposase proteins!
• They then move back into the nucleus!
LINE transposition
Ostertag et al., 2007. Genome Biology 8: S16
• The transposase proteins catalyse the integration of the LINE into a new target site, using a mechanism called “target-primed reverse transcription”!
Target-primed reverse transcription
• The endonuclease cuts the target site, which has a short region that can anneal to the poly-A tail!
• The LINE RNA anneals to the single-strand DNA here, and reverse transcriptase starts synthesizing DNA using the LINE RNA as the template!
• A second (staggered) cut is made in the target site, and the newly-synthesized DNA joins onto this!
• DNA polymerase then replaces the LINE RNA with DNA, therefore completing insertion of the LINE at the new site with target site duplications!
• Truncated copies occur predominantly at the 5’ end! biol.lf1.cuni.cz/ucebnice/en/repetitive_dna.htm
SINEs
• They are 100-300 bp in size!• All SINEs are transcribed into RNA, and are believed to
use the LINE transposase machinery to catalyse their transposition; therefore their transposition mechanism is the same as that for LINEs!
• They make target-site duplications!
Prak & Kazazian, Nature Reviews Genetics, 2000, 1: 134-
• Almost all SINEs (apart from Alu) are derived from tRNA genes!
• They contain a 3’ UTR region that is related to LINEs, and also have a poly-A tail!
Alus
• Most abundant SINE in the human genome is called Alu (have an AluI restriction site)!
• Derived from a small RNA called 7SL RNA; SINE RNA forms a distinct secondary structure, similar to 7SL RNA!
biol.lf1.cuni.cz/ucebnice/en/repetitive_dna.htm
LTRs
• LTR = long terminal repeats; these have long direct repeats (100 bp - several kb) at each end!
• Contains gag and pol genes; the pol protein contains reverse transcriptase, RNase, and integrase activities!
• Related to retroviruses; difference is presence of an env gene (allows production of the viral particle) and the prt gene!
• Mechanism of transposition: recombination of the cDNA into the genome using the LTRs!
• Makes target site duplications, but very short!• No known autonomous LTRs in the human genome!
biol.lf1.cuni.cz/ucebnice/en/repetitive_dna.htm
Effects on the genome - mutation
• Transposons cause mutations by inserting into genes and regulatory sequences, etc.!
• Even if the transposon is a conservative transposon, it can leave behind target site duplications, which are a form of mutation!
Hartwell, Genetics: From Genes to Genomes, 2008
Effects on the genome - rearrangement
• Transposition with two nearby transposons can move intervening genes; can duplicate or delete these genes!
• Recombination between transposons at different sites can cause duplications & deletions!
• This is dangerous with transposons on different chromosomes; causes genomic instability!
Hartwell, Genetics: From Genes to Genomes, 2008
Exon duplication via SINE mobilization
• SINE transcription can extend past the normal stop signal!
• Reinsertion occurs elsewhere in the genome!• Reverse transcription duplicates both the
SINE and exon 2!
exon 1 SINE
intron
exon 2
SINE transcript SINE exon 2
Effects on the genome - duplication
Effects on the genome – summary I
Prak & Kazazian, Nature Reviews Genetics, 2000, 1: 134-
• Insertion mutation!
• Target site mutation!
• Movement of genes!
• Recombination duplicates or deletes genes; genomic instability!
• Deletion or duplication of genes between transposons!
• The cell tries to control transposition, e.g. siRNA!
Effects on the genome – summary II
• In addition, the double-strand breaks introduced by transposons can cause mutations!
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