1. Transposible elements Viruses and viroids

2. Transposons, TE = mobile genetic elements -sequences of DNA that can move around to different positions within the genome of a single cell (transposition), -cause mutations and chromosomal rearrangements -identified in Prokaryotes and all Eukaryotes: (with exception of parasitic Plasmodium falciparum) animals 3-45%, fungi 2-20%, plants 10-80% in plants: - thousands of families, form majority of repetitive DNA

3. Basic classification of TE DNA transposons - „CUT and PASTE“ (rarely „COPY and PASTE“) typically: transposase cleaves out, inserts to new site Retrotransposons - „COPY and PASTE“ typically: - reverse transcriptase - DNA copies from TE transcripts - integrase - insertion

4. Basic classification of TE - self-sufficiency Autonomous elements – encode genes necessary for transposition/ replication Non-autonomous – derivatives of autonomous elements – lost of genes for transposition/replication – keep sequences necessary for transposition (can be mobilized by related autonomous elements!)

5. Discovery of transposons Barbara McClintock (1902-1992) Nobel prize in Physiology and Medicine 1983 Mobile genetic elements in maize 1940-1950

6. Discovery of TE - study of chromosomal breakage - increased frequency in certain site (= marker „dissociation“ Ds) - location of Ds was unstable after crossing with some lines (= line carrying „activator“ Ac)

7. - in one location – Ds insertion was connected with loss of purple pigment of endosperm - after crossing with activator line pigment synthesis was recovered in some cells Discovery of TE

8. Barbara McClintock (1902-1992) 1951: formulated basic context of epigenetics: "[T]he progeny of two (such) sister cells are not alike with respect to the types of gene alteration that will occur. Differential mitoses also produce the alterations that allow particular genes to be reactive. Other genes, although present, may remain inactive. This inactivity or suppression is considered to occur because the genes are ‘covered' by other nongenic chromatin materials. Gene activity may be possible only when a physical change in this covering material allows the reactive components of the gene to be ‘exposed' and thus capable of functioning. "

9. Nature Rev. Genet., 2007 1.Class: - replication with/without RNA intermediate DNA transposons Retrotransposons 2.Subclass: - mechanism of replication (DNA transposones) 3.Order: - basic structural features 4.Superfamily: - similarity of sequences Classification of TE

10. Basic TE subclasses Lisch 2013, Nature Rev. Genet. /replication protein + helicase

11. Class II: DNA transposons

12. DNA transposons - subclass I transposition: break, religation - terminal inverted repeats recognized by transposase (fungal TE Crypton encodes recombinase instead of transposase) - duplication of short seq. (2-8 bp) = footprint after transposition - clustering in genom, hundreds of copies

13. Multiplication of DNA transposons + break repair by homologous recombination (TE can be restored) Activation during replication - how?Hemimethylated state?

14. Examples of DNA transposons – subclass I: - Ac, Spm, Mu (maize), Tam (Antirrhinum), TphI (petunia), TagI (Arabidopsis) - non-autonomous: Ac/Ds, Spm/dSpm - Stowaway, Tourist >10 000 copies

15. Helitrons – single strand breake and strand displacement - Rep/helikase-like, replication protein A-like - maize: 4 - 10 000 gene fragments mobilized by helitrons DNA transposons – subclass 2

16. Class I: Retrotransposons

17. Retrotransposons - replication through RNA intermediate (multiple offspring) - related to retroviruses - millions of copies - huge portions of genome (up to 40-80 % of genome size) - element size 1-13 kbp Order LTR – most important in plants - LTR (long terminal repeat) - promotor, terminator, direct repeat - dubling of short target sequence - gag (nucleocapsid), - pol (protease, reverse transcriptase-RNase H, integrase)

18. Retrotransposons LTR - replication - LTR (U3, R, U5) - PBS tRNA primer -skips between templates (direct repeat)

19. Examples of LTR retrotransposons BARE-1, barley, 12,1 kbp, >50 000 copies, transcript in leaves and callus PREM-2, maize, 9,5 kbp, >10 000 copies, transcript in microspores Tnt1, tobacco, 5,3 kbp, >100, activated after wounding, patogen attack, Ty3 – gypsy group – ancestors of Caulimoviruses, hypothetical ancestors of animal retroviruses (env-like sequence) Athila, A.t., 10,5 kbp, >10000, paracentromeric regions

20. Retrotransposons without LTR LINE (long interspersed nuclear elements) SINE (short interspersed nuclear elements) LINE - phylogenetically most original, ancestors of LTR - 5´region – promoter; 3´ region - terminator Cin4, maize, 1-6,8kbp, 50-100, various truncated forms SINE - non-autonomous – use RT of other elements - derived from products of RNA polymerase III (tRNA, 7SLRNA, (rRNA)) - < 500 nt LINE APE – endonuklease, RH – RNase H

21. Regulation of transposon activity - both endogenous and by plant cell - mostly inactive – methylated (prevents activity and also illegitime recombination (crossing over) - often developmentally regulated activation - rarely activation by environmental conditions: Tam1 (1000x at 15°C) Reme1– activation by UV light

22. Zemach et al. 2013 Maintenance methylation of TE

23. Role of TE in evolution causing mutations = increasing variability - modulation of expression (activation, repression, developmentally- or stress-induced) – important during domestication (WHY?) - new gene evolution - genome evolution - in plants no direct profit – no genes directly increasing fitness (like resistances in bacteria) - increase in fitness by random mutation – low probability, but possible (really occuring)

24. Transposon-mediated mutagenesis - site of insertion - character of transposon regulatory sequences - modulation of transcription (spatial, temporal) – promoter, enhancers - transcript stability and splicing - changes in protein sequence (footprints, frame-shift) – possible role in evolution of new genes Promotor 5´UTR exon intron exon 3´UTR terminator

25. TE affected expression of TF VvmybA1 - regulation of antokyan synthesis genes (Kobayashi et al. 2004, Science)

26. TE affected gene expression - examples Maize: – inactivation of CCT (photoperiod response) by CACTA-like element (DNA TE) insertion to promoter – allowed cultivation in temporal climate (long-day flowering) block of branching (TE enhancer  OE of inhibitor) (Yang et al. 2013, PNAS) (Butelli et al. 2012, Plant Cell) Red orange: Ruby – myb TF (regulation of antokyan genes) activated by TE insertion - cold induced expression in fruits

27. Gene for transposase (inducible expression) Ds Transposon mutagenesis - mainly in Arabidopsis - insertional mutagenesis (alternative to T-DNA mutagenesis – see later) – easy detection of the site of insertion (x chemical mutagenesis) - DNA transposons from maize – low frequency of transposition - two-component systém (transposase/Ds) Line with non-autonomous element in resistance gene R Selection of resistant plants = with transposed transposon Selection in next for plants without transposase gene DsR - frequent mutagenesis of near genes – clustering (20 % in 1Mb around) - reintroduction of transposase – possibility to reverse the mutation

28. Role in gene evolution - insertional mutagenesis (premature termination), footprints - participation in gene duplications - directly or via recombination (TE = homologous repeats) - formation of intron-less gene copies (reverse transcription) - genes of TE origin „domesticated“ by many eucaryotic organisms for new functions (telomerase, syncitin, ….) - new gene formation by fusions of mobilized gene fragments (helitrons)

29. Role in genome evolution Chromosomal rearrangements - changes of linkage groups - speciation (incompatibility) Increase in genome size („genomic obesity“) multiplication of TE x homologous recombina tion can prevent or even decrease genome size (2n cottons – 2 -3x genome size differences by active recombination; Hawkins 2009 PNAS )