Мобильные элементы и эволюция генома М.Б. Евгеньев, Е.С. Зеленцова, Н.Г. Шостак, Е.Н. Мяснянкина,

1. Мобильные элементы и эволюция генома М.Б. Евгеньев, Е.С. Зеленцова, Н.Г. Шостак, Е.Н. Мяснянкина, О.Г. Зацепина, Д.Г. Гарбуз, Шилова В. Ю. и М.И. Соколова Институт Молекулярной Биологии им. В.А. Энгельгардта, РАН

6. Genome invasion or de novo formation Dynamics of invasion Host defense Multiplication Regulation Evolution Genetic burden Variability Deleterious effect Death Domestication Loss Maintenance Distribution in sequenced genomes

7. Ectopic recombination Low recombination rate region Heterochromatin Low expression level region Low gene density region TE-products poisoning Gene-disruption

11. Gross morphology: ovaries Bilateral and unilateral sterile ovaries in dysgenic D.virilis

12. Ovariole morphology:agametic ( in dysgenic ovaries)

13. Drosophila virilis adult dysgenic testis

14. Forming the primordial gonads in Drosophila virilis dysgenic 9 strain - wild type - control

15. Reduction in pole cells in dysgenic embryos, cntd

19. Drosophila virilis: whole mount hybridization with Penelope DNA probe 160 9 160x9 9x160

22. intron GT AG AT TG 5' cDNA fragment Penelope transcript XhoI EcoRI BamHI EcoRI XhoI 1 kb. Penelope, clone p6 Schematic representation of Penelope clone isolated from the genome of D.virilis. 5’fragment of cDNA with intron 75 b.p. length is shown above

23. A ov+ th± D ov± th+ B ov+ th± F ov- th+ N ov- th+ O ov- th+ C ov+ th± P ov- th+ R ov- th- G ov- th- ∆ (416 – 491) S ov- th- 371 Intron ORF Penelope 416 491 1 250 302 369 416 487 544 570 611 680 850 371 Regulatory elements - transcription start Promoter

24. Neptune 100 Penelope Penelope branch 93 Xena 100 47 Poseidon Tel H.sap Telomerases 96 Tel S.pom retr S.ent Ec79 99 100 Retrons 40 Ec110 88 Ec86 94 62 Ec67 Mat S.aga 100 Mat N.aro Maturases 99 61 Cox S.cer 100 Mat S.cer RT T.cru 90 TART-B2 59 RT L.pol Non-LTR retrortansposons 100 RT A.mar 69 RT R2Dm 94 RT R2Nvi Md M.mus 71 LTR retrortansposons RT C.alb Cre 2 79 HIV 1 Neighbor-joining tree based on the multiple alignment of 7 conserved motifs of reverse transcriptase domain Penelope – retrotransposon from D.virilis; Neptune – retrotransposon from Fugu rubripes; Poseidon - retrotransposon from Tetraodonnigroviridis; Xena - retrotransposon from Takifugu rubripes

25. Fig.Multiple alignment of the C-terminal region of Penelope with a selection of GIY-YIG domain endonucleases. * shows the positions of conservative AA residues selected for mutagenesis of Penelone endonuclese catalytic domain. Substituted AA residues are shown above (Y736H, Y750H, R761A, H782G, D793A, E801G).

26. 1 2 3 4 5 kDa 220 97 67 46 30 20 Purification of Penelope protein from Spodoptera frugiperda cells infected by baculovirus containing Penelope ORF Penelope protein was purified by multiple step chromatography using: 2) - phosphocellulose P11 (Whatman); 3) – heparin-sepharose (Pharmacia); 4) - MonoS (FPLC, Pharmacia) and 5) - concentrated using 50 kDa Centricon (Millipore)

27. Изучение влияния мутаций по rasiRNA-зависимому пути на количество транскриптов мобильного элемента Рenelopeс использованием метода RT-PCR Количество транскриптов мобильного элемента Penelopeна фоне мутации spn-e spn-e Pen +/- -/- уровень экспрессии Pen rp49 Количество транскриптов мобильного элемента Penelopeна фоне мутации armitage armi Pen +/- +/+ -/- Pen уровень экспрессии rp49

29. Table 1. Phylum Class Species PLEs 10 18 about 100 Penelope, Athena, (e.g. Protista, Rotifera, (e.g. Crustacea, Amphibia, Cercyon, Bridge, Arthropoda, Chordata) Echinoidea, Insecta) Xena

30. H S X E K E X E H pUC-8 S S A Penelopep6 white 3'P 5'P H S H pUC-8 S S X X E K E K S B white Penelope ORF Pr 5'P 3'P pUC-8 H X E K X H S S S S C white 3'P PenORF Pr 5'P 1kb Schematic representation of the structure of Penelope copies integrated into D. melanogaster genome DNA construction containing A) full length Penelope clone p6; B) full length Penelope ORF under control heat shock promoter of D.melanogaster; C) Penelope ORF with a deletion of 5' region under control heat shock promoter of D.melanogaster. Arrows with 3'P and 5'P mean 3'P and 5'P inverted repeats of P element, respectively. Arrow with Pr displays a Hsp70Bb heat shock promoter of D.melanogaster. The symbols indicated above correspond the restriction sites (X, H, E, S and K represent XhoI, HindIII, EcoRI, SalI and KpnI, respectively)

32. H S X X H S S pUC-8 Penelopep6 white 3'P 5'P 8,5 kb kb. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10.0 8.0 6.0 5.0 4.0 3.0 2.5 2.0 1.5 1.0 Southern blot hybridization of transformed D.melanogaster strains with Penelope probe Lanes 1-3 initial strain Df(I)w, yw67c23(2); lanes 4-6; 7-9 strains transformed with Penelope ORF under control of heat shock promoter; 10-12; 13-15; strains transformed with full length Penelope copy (p6). Lanes 1- 4; 7; 10; 13 genomic DNA digested by XhoI; lanes 2; 5; 8; 11; 14 genomic DNA digested by Hind III; lanes 3; 6; 9; 12; 15 genomic DNA digested by Sal I

33. А B Results of in situ hybridization of polytene chromosomes of flies D.melanogaster transformed by Penelope-containing clone (p6) А - in situ hybridization with "mini-white"; B - hybridisation with Penelope. Hybridization sites are shown by arrows

34. Results of in situ hybridization of polytene chromosomes of flies D.melanogaster transformed by Penelope-containing clone (p6)

36. Drosophila melanogaster transformation with the Penelope transposable element Sterile ovaries Agametic ovarioles

37. The design of localization of EPgy2 insertions into hsp70Aa and hsp70Ab genes in strains with US-4 and US-2. Number of primers is indicated. EPgy2 6 5 7 5 3 1 2 4 54 CG18347 hsp70Aahsp70Ab CG3281 aur 53 CG12213 4 2 1 3 55 5 6 7 5 EPgy2

38. P-element construct hsp70Aa hsp70Ab

39. EPgy2 ∆2-3 ∆2-3 Yw P(y+w+) ♀ X X ♂ Yw P(y+w+) yw yw P(y+w+) yw + ♀ Df X ♂ ∆2-3 yw + yw yw P(y+w+) + ♀ Df X yw ♂ + + n P(y+w+) P(y+w+) + Selection of males with enhanced eye color n n P(y+w+) P(y+w+) P(y+w+) P(y+w+) yw yw ♀ X ♂ yw + + Selection of strains, containing additional copies of construction

40. Southern-hybridization of genome DNA from heterozygous transformants with 5’ (up) and 3’ (down) fragments of D. melanogaster hsp70 (HindIII-BamHI) 4.3 kb Bbb 3.3 kb Bb+Bc 2.5 kb Aa 2.1 kb Ba 1.8 kb Ab 4.4 Bc 4.3 Ab, Bb 2.0 Aa, Ba

41. 57% Strains from US-2 hsp70Aa hsp70Ab 43% -229(1) 200(1) -135(4) -97(3) +1 H H H T H g g g g g -160(1) -96(3) -40(1)

42. 87% Strains from US-4 hsp70Aa hsp70Ab 13% CGGCGCAC target duplication -137(1) -240(1) -174(1) -135(4) -97(12) -42(2) -28(1) +1 H H H T H g g g g g -144(1) -96(5) -40(1) GGCGCACT target duplication

43. Сравнение экспериментальной и естественной транспозиции

44. ВЫВОДЫ • МОБИЛЬНЫЕ ЭЛЕМЕНТЫ СОСТАВЛЯЮТ ЗНАЧИТЕЛЬНУЮ ПРОПОРЦИЮ ГЕНОМА ВСЕХ ИЗУЧЕННЫХ ОРГАНИЗМОВ. • ПРИ ПРОВЕДЕНИИ ОПРЕДЕЛЕННЫХ СКРЕЩИВАНИЙ ПРОИСХОДИТ АМПЛИФИКАЦИЯ И МАССОВЫЕ ТРАНСПОЗИЦИИ ОПРЕДЕЛЁННЫХ СЕМЕЙСТВ МОБИЛЬНЫХ ЭЛЕМЕНТОВ («СИНДРОМ ГИБРИДНОГО ДИСГЕНЕЗА»). • ВЫСОКИЙ УРОВЕНЬ ГЕНЕТИЧЕСКОГО ПОЛИМОРФИЗМА, ВОЗНИКАЮЩИЙ ПРИ СИНДРОМ ГИБРИДНОГО ДИСГЕНЕЗА, МОЖЕТ СЛУЖИТЬ МАТЕРИАЛОМ ДЛЯ ОТБОРА, ПРИВОДЯ К БЫСТРОМУ, «ВЗРЫВНОМУ» ВИДООБРАЗОВАНИЮ.