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 Evolution Multiplication Domestication Loss Maintenance Host defense Genetic burden Deleterious effect Regulation Variability Death Distribution in sequenced genomes Dynamics of invasion

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

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 160x99x160 160 9

22. Penelope transcript GT 5' cDNA fragment intron AG AT TG 1 kb. Penelope, clone p6 XhoIEcoRI BamHI XhoI 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. 371 Intron 1 250 302 369 416 487 544 570 611 680 850 S ov- th- R ov- th- 371 P ov- th+ O ov- th+ N ov- th+ F ov- th+ B ov+ th± C ov+ th± D ov± th+ A ov+ th± 416 491 Promoter Regulatory elements G ov- th- ∆ (416 – 491) - transcription start ORF Penelope

24. 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 Tetraodon nigroviridis; 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. kDa 220 97 67 46 30 20 1 2 3 4 5 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 Рenelope с использованием метода RT-PCR Pen +/- -/- spn-e уровень экспрессии Количество транскриптов мобильного элемента Penelope на фоне мутации spn-e Pen rp49 Pen +/- +/+ -/- armi Pen rp49 уровень экспрессии Количество транскриптов мобильного элемента Penelope на фоне мутации armitage

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. pUC-8 1kb Penelope p6 KXE white H S S E E X 3'P H S Penelope ORF KXEE 3'P Pr K X 5'P white H S S S Pen  ORF X S K E 3'P Pr X 5'P white H S S A B C H S 5'P 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. kb. 10.0 8.0 6.0 5.0 4.0 3.0 2.5 2.0 1.5 1.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Southern blot hybridization of transformed D.melanogaster strains with Penelope probe Lanes 1-3 initial strain Df(I)w, yw 67c23(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 pUC-8 Penelope p6 X white H S S X 3'P H S 5'P 8,5 kb

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. hsp70Aa hsp70Ab CG3281 aur CG12213 EPgy2 3 1 2 4 54 4 2 1 3 55 6 7 5 5 CG18347 EPgy2 53 5 5 6 7

38. P-element construct hsp70Aahsp70Ab

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

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

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.