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

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

TE-products poisoning

Gene-disruption

Ectopic recombination

Low recombination

rate region

Low expression level region

Low gene density region

Heterochromatin

Gross morphology: ovariesGross morphology: ovaries

Bilateral and Bilateral and unilateral unilateral

sterile ovaries sterile ovaries in dysgenic in dysgenic

D.virilisD.virilis

Ovariole morphology:Ovariole morphology:agametic agametic

( in dysgenic ovaries)( in dysgenic ovaries)

Drosophila Drosophila virilisvirilis adult adult dysgenic dysgenic

testistestis

Forming the primordial gonads in Forming the primordial gonads in Drosophila virilisDrosophila virilis

dysgenicdysgenic 9 strain - wild type - control9 strain - wild type - control

Reduction in pole cells in Reduction in pole cells in dysgenic embryos, cntddysgenic embryos, cntd

Drosophila virilisDrosophila virilis: whole mount : whole mount hybridization with hybridization with PenelopePenelope DNA DNA

probeprobe

160x9 9x160

160

9

Penelope transcript

GT

5' cDNA fragment

intron AG

AT TG

1 kb.

Penelope, clone p6

XhoI EcoRI EcoRIBamHI 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

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

Regulatoryelements

Gov- th-

∆(416 – 491)

- transcription start

ORF Penelope

.2

.4

.6

.8

1 .0

Neptune

Penelope

Xena

Poseidon100

93

100

Tel H.sap

Tel S.pom96

47

retr S.ent

Ec79

Ec110100

Ec86

Ec6794

88

99

Mat S.aga

Mat N.aro

Cox S.cer

Mat S.cer100

99

100

62

RT T.cru

TART-B2

RT L.pol

RT A.mar

RT R2Dm

RT R2Nvi94

69

100

Md M.mus

RT C.alb71

59

90

61

40

Cre 2

HIV 179

Penelope branch

Telomerases

Retrons

Maturases

Non-LTR retrortansposons

LTR retrortansposons

Neighbor-joining tree based on the multiple alignment of 7 conserved motifs of reverse transcriptase domainPenelope – retrotransposon from D.virilis; Neptune – retrotransposon from Fugu rubripes; Poseidon - retrotransposon from Tetraodon nigroviridis; Xena - retrotransposon from Takifugu rubripes

H H A G A G * * * * * *1224056 Penelope 734 VVYQIPC4KC-NSVYIGTTKS---KLKTRISQHKSDFKLRHQ--NNIQKTALMTHCIRSNHTP-NFDETTI---------------LQQEQHYN 805

1181450 PBCV1 6 YFYIWIHKIS-GEKYVGITTQ---EIQQRLKAHCRKGNKC-----RRLRNAIQKHGVDAFD----VEYFEWC---------DPWDLAYIEGLLI 772738435 ChiV 5 YIYVIENNFD-NHVYIGSTVD---SLENRFRRHKADALKRPSC---LFHTYMKKHGVDNFVI-KLLKEVEII---------SILDLHLLEQNFI 813033368 coliphage T4 4 GIYQIKNTLN-NKVYVGSAKD----FEKRWKRHFKDLEKGCHS-SIKLQRSFNKHG-NVFEC-SILEEIPYE----------KDLIIERENFWI 792492912 MJ0613_Mj 258 GKTERFFKKG-YYFYIGSAFGNSMNLKNRIERHLKD------------DKKMHWHIDYLLKY-GKIEEIYIT-------------NERVECEVA 3242621722 endoIII_Mth 23 SLGTVRFPPG-FYVYVGSGFSS---LEARIKRHLSS------------EKRRRWHIDHFLDG-AEVECVLYT-----------TDKRRLECAVS 882650260 endoIII_Af 3 ALGVIAFRRG-YYYYVGSANS----GVHRVKRHFSI------------KKKKRWHIDYISAK-MEVVGAILS--------------KEPECGLA 64586345 YB78_YEAST 15 CCYLLQSINKRQSFYVGSTPN----PVRRLRQHNGKLA---------VGGAYRTKRDGSR----PWEMIMIV-----RGFPSKIAALQFEHAWQ 861175105 Y079_NPVAC 12 CVYILRQDNG--KLYTGITSN----LNRRIKQHSNK------------QGAKCLRNAT------NLRLVYHS-----ASAYDYNTAARMEYNLK 761176177 YHBQ_Ec 5 FLYLIRTADN--KLYTGITTD----VERRYQQHQSG------------KGAKALRGKG------ELTLVFSA------PVGDRSLALRAEYRVK 682635759 yurQ_Bs 45 GIFMFYNIHD-ELLFVGKARK----LRQRIKKHFEDT-------------VSPIKHHRDEVY--KIEVCVVD--------------DPMEREIY 1042621507 UvrC_Mt 15 GVYIFRDRDD-RVLYVGKSIS----IRKRVSSYFREQ------------ENPRLRIMMRHLE--SIEYILTQ--------------NEKEALIL 751174919 UvrC_Ec 19 GVYRMYDAGG-TVIYVGKAKD----LKKRLSSYFRSN-----------LASRKTEALVAQIQ--QIDVTVTH--------------TETEALLL 801770045 UvrC_Bs 17 GCYLMKDRQQ-TVIYVGKAKV----LKNRVRSYFTGS------------HDAKTQRLVTEIE--DFEYIVTS--------------SNLEALIL 7714037 Intron Pa 18 GIYMWTNKLN-GKKYVGSSVD----LRRRLSEYYNINRILNEK--SMPINVALLKYGYTNFTLTILEICDKD------------SLMSREKHFF 922738528 Intron Aa 98 GVYCLINKIN-GNAYVGSSIN----LASRMKNYLNNIFLKSKKNINMPIVKALLKYNQESFTLLILEYVEPN------------YLTIRETYYI 1741236427 Intron Am 75 GIYGIINNKT-NKIYIGSAVN----LHKRLVEHLYSDKT------NIRLQRSIGKDGLSSFSFIIFEYHDFN-DSILEFIDFNDLLLESETFYI 1562865254 ORF_Ce 84 GVYLIYDNLT-HDFYVGSAIT--NRINVRFRNHCIHKSG------SSLVAKAIQKSGLENFDFYILEYFHGFVHKENQKKDQLE-LLKRETYYL 167

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).

kDa220

97

67

46

30

20

1 2 3 4 5

Purification of Penelope protein from Spodoptera

frugiperda cells infected by baculovirus containing

Penelope ORFPenelope 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)

Изучение влияния мутаций по Изучение влияния мутаций по rasiRNArasiRNA--зависимому пути зависимому пути на количество транскриптов мобильного элемента на количество транскриптов мобильного элемента

РРenelopeenelope с использованием метода с использованием метода RT-PCRRT-PCR

-/- +/-

Penelope(A1)

0

2

4

6

8

10

12

14

16

18

-/-

+/-

Penelope (1)

Pen +/- -/-

spn-e

уров

ень

эксп

ресс

ии

Количество транскриптов мобильного элемента Penelope на фоне мутации spn-e

Pen

rp49

Pen +/- +/+ -/-

armi

Pen

rp49 +/+ +/--/-

Penelope(A1)

02468

10121416

-/-

+/+

+/-

Penelope (1)

уров

ень

эксп

ресс

ииКоличество транскриптов мобильного элемента

Penelope на фоне мутации armitage

  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

pUC-8

pUC-8

pUC-8

1kb

Penelope p6

K X E

white

H

S S EEX

3'P

H S

Penelope ORF

KX E E

3'P Pr

K X

5'Pwhite

H

S S S

PenORF

X S K E

3'P Pr

X

5'Pwhite

H S S

A

B

C

H S

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)

kb.

10.0 8.0 6.0 5.0 4.03.0

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, 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

pUC-8Penelope p6

X

white

H

S S X

3'P

H S

5'P

8,5 kb

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

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

StrainSites of Original

Construct InsertionNumber ofPenelope

transposition

All sites wherePenelope insertions

were foundStrain AEstablished inOctober, 1999

3B (Ich.);18A (Ich.)

_

Tested March,2000

3B (Ich.);18A (Ich.)

4 21C, 6E, 54F, 100F

TestedSeptember, 2000 3B (Ich.);

18A (Ich.)9 (new)

22F, 70D, 100B,55A, 54A, 28E, 95E,56C, 92D(13 total)

Strain BEstablished inOctober, 1999

6E (Ich.);96F (IIIR ch.)

_

TestedSeptember, 2000

6E (Ich.);96F (IIIR ch.)

5 17F, 24A, 51B, 55F,57B (5 total)

ch – means chromosoma

Drosophila melanogasterDrosophila melanogaster transformation with the transformation with the PenelopePenelope

transposable elementtransposable element

Sterile ovaries

Agametic ovarioles

The design of localization of EPgy2 insertions into hsp70Aa and hsp70Ab genesin 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

75

5

CG18347

EPgy2

53

5

5

6

7

P-element construct

hsp70Aa hsp70Ab

X

EPgy2 ∆2-3

♀ Df

♂

yw

yw

yw

∆2-3

P(y+w+) X

++

ywyw♀

Df

++ X

♂

Selection of males with enhanced eye color

yw P(y+w+)

+

+

P(y+w+) P(y+w+) n

♀++ X

P(y+w+) P(y+w+) nyw yw P(y+w+) P(y+w+)n

yw

X

Selection of strains, containing additional copies of construction

♂

Yw P(y+w+)

Yw P(y+w+) yw

∆2-3♀

♂

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 Ba1.8 kb Ab

4.4 Bc4.3 Ab, Bb

2.0 Aa, Ba

hsp70Aa hsp70Ab

Strains from US-2 57%

43%

H H H T+1

-229(1) 200(1) -135(4) -97(3)

-160(1) -96(3) -40(1)

Hg g g g g

GGCGCACT target duplication

H H H T+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)

Hg g g g g

Сравнение экспериментальной и естественной транспозицииСравнение экспериментальной и естественной транспозиции

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