Мобильные элементы и эволюция генома

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
Dynamics
of
invasion
Multiplication
Evolution
Host defense
Regulation
Variability
Genetic burden
Deleterious effect
Death
Domestication
Loss
Maintenance
Distribution in sequenced genomes
Ectopic
recombination
Low
recombination
rate region
Heterochromatin
Low
expression
level region
TE-products
poisoning
Low gene
density
region
Genedisruption
Gross morphology: ovaries
Bilateral and
unilateral
sterile ovaries
in dysgenic
D.virilis
Ovariole morphology:
agametic
( in dysgenic ovaries)
Drosophila
virilis adult
dysgenic
testis
Forming the primordial gonads in
Drosophila virilis
dysgenic
9 strain - wild type - control
Reduction in pole cells in
dysgenic embryos, cntd
Drosophila virilis: whole mount
hybridization with Penelope DNA
probe
160
9
160x9
9x160
GT
intron
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
A ov+ th±
D ov± th+
B ov+ th±
F ov- th+
N ov- th+
O ov- th+
C ov+ th±
P ov- th+
1.0
.8
R ov- thS ov- th371
250 302
.4
(416 – 491)
416
1
.6
G
ov- th∆
369 416
371
.2
Intron 491
ORF Penelope
487 544 570 611
680
Regulatory
elements
- transcription start
Promoter
850
Neptune
100
93
Penelope
Penelope
Xena
branch
100
47
Poseidon
96
Tel H.sap
Tel S.pom
Telomerases
retr S.ent
Ec79
99
100
40
Ec110
88
94
62
Retrons
Ec86
Ec67
Mat S.aga
100
Mat N.aro
99
61
Maturases
Cox S.cer
100
Mat S.cer
RT T.cru
90
TART-B2
59
RT L.pol
100
RT A.mar
69
94
Non-LTR
retrortansposons
RT R2Dm
RT R2Nvi
71
Md M.mus
RT C.alb
79
LTR retrortansposons
Cre 2
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 Tetraodon nigroviridis; Xena retrotransposon from Takifugu rubripes
1224056 Penelope
1181450
2738435
3033368
2492912
2621722
2650260
586345
1175105
1176177
2635759
2621507
1174919
1770045
14037
2738528
1236427
2865254
H
H
A
G
A
G
*
*
*
*
*
*
734 VVYQIPC4KC-NSVYIGTTKS---KLKTRISQHKSDFKLRHQ--NNIQKTALMTHCIRSNHTP-NFDETTI---------------LQQEQHYN 805
PBCV1
6
ChiV
5
coliphage T4 4
MJ0613_Mj
258
endoIII_Mth 23
endoIII_Af
3
YB78_YEAST
15
Y079_NPVAC
12
YHBQ_Ec
5
yurQ_Bs
45
UvrC_Mt
15
UvrC_Ec
19
UvrC_Bs
17
Intron Pa
18
Intron Aa
98
Intron Am
75
ORF_Ce
84
YFYIWIHKIS-GEKYVGITTQ---EIQQRLKAHCRKGNKC-----RRLRNAIQKHGVDAFD----VEYFEWC---------DPWDLAYIEGLLI
YIYVIENNFD-NHVYIGSTVD---SLENRFRRHKADALKRPSC---LFHTYMKKHGVDNFVI-KLLKEVEII---------SILDLHLLEQNFI
GIYQIKNTLN-NKVYVGSAKD----FEKRWKRHFKDLEKGCHS-SIKLQRSFNKHG-NVFEC-SILEEIPYE----------KDLIIERENFWI
GKTERFFKKG-YYFYIGSAFGNSMNLKNRIERHLKD------------DKKMHWHIDYLLKY-GKIEEIYIT-------------NERVECEVA
SLGTVRFPPG-FYVYVGSGFSS---LEARIKRHLSS------------EKRRRWHIDHFLDG-AEVECVLYT-----------TDKRRLECAVS
ALGVIAFRRG-YYYYVGSANS----GVHRVKRHFSI------------KKKKRWHIDYISAK-MEVVGAILS--------------KEPECGLA
CCYLLQSINKRQSFYVGSTPN----PVRRLRQHNGKLA---------VGGAYRTKRDGSR----PWEMIMIV-----RGFPSKIAALQFEHAWQ
CVYILRQDNG--KLYTGITSN----LNRRIKQHSNK------------QGAKCLRNAT------NLRLVYHS-----ASAYDYNTAARMEYNLK
FLYLIRTADN--KLYTGITTD----VERRYQQHQSG------------KGAKALRGKG------ELTLVFSA------PVGDRSLALRAEYRVK
GIFMFYNIHD-ELLFVGKARK----LRQRIKKHFEDT-------------VSPIKHHRDEVY--KIEVCVVD--------------DPMEREIY
GVYIFRDRDD-RVLYVGKSIS----IRKRVSSYFREQ------------ENPRLRIMMRHLE--SIEYILTQ--------------NEKEALIL
GVYRMYDAGG-TVIYVGKAKD----LKKRLSSYFRSN-----------LASRKTEALVAQIQ--QIDVTVTH--------------TETEALLL
GCYLMKDRQQ-TVIYVGKAKV----LKNRVRSYFTGS------------HDAKTQRLVTEIE--DFEYIVTS--------------SNLEALIL
GIYMWTNKLN-GKKYVGSSVD----LRRRLSEYYNINRILNEK--SMPINVALLKYGYTNFTLTILEICDKD------------SLMSREKHFF
GVYCLINKIN-GNAYVGSSIN----LASRMKNYLNNIFLKSKKNINMPIVKALLKYNQESFTLLILEYVEPN------------YLTIRETYYI
GIYGIINNKT-NKIYIGSAVN----LHKRLVEHLYSDKT------NIRLQRSIGKDGLSSFSFIIFEYHDFN-DSILEFIDFNDLLLESETFYI
GVYLIYDNLT-HDFYVGSAIT--NRINVRFRNHCIHKSG------SSLVAKAIQKSGLENFDFYILEYFHGFVHKENQKKDQLE-LLKRETYYL
77
81
79
324
88
64
86
76
68
104
75
80
77
92
174
156
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).
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)
Изучение влияния мутаций по rasiRNA-зависимому пути
на количество транскриптов мобильного элемента
Рenelope с использованием метода RT-PCR
-/-
Pen
rp49
armi
Pen +/- +/+ -/Pen
rp49
18
Penelope(A1)
16
14
12
-/+/Penelope (1)
10
8
6
4
-/-
2
+/-
0
Количество транскриптов мобильного элемента
Penelope на фоне мутации armitage
уровень экспрессии
spn-e
Pen +/-
уровень экспрессии
Количество транскриптов мобильного элемента
Penelope на фоне мутации spn-e
16
14
12
10
8
6
4
2
0
Penelope(A1)
-/+/+
+/Penelope (1)
-/-
+/+
+/-
Table 1.
Phylum
10
Class
Species
18
about 100
(e.g. Protista, Rotifera, (e.g. Crustacea, Amphibia,
Arthropoda, Chordata)
Echinoidea, Insecta)
PLEs
Penelope, Athena,
Cercyon, Bridge,
Xena
pUC-8
K
E
K
S
E
white
X
5'P
H
S
pUC-8
E
PenORF
K
X
Pr
S
S
3'P
X
white
Pr
white
S
Penelope ORF
3'P
HS
C
X
H
S
B
XE
E
Penelope p6
3'P
HS
K
E
S
A
X
S
HS
5'P
H
pUC-8
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)
S
X
X
H
S
HS
pUC-8
3'P
Penelope p6
white
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
B
А
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)
Strain
Strain A
Established in
October, 1999
Tested March,
2000
Tested
September, 2000
Sites of Original
Construct Insertion
Number of
Penelope
transposition
3B (Ich.);
18A (Ich.)
3B (Ich.);
18A (Ich.)
_
3B (Ich.);
18A (Ich.)
Strain B
Established in
6E (Ich.);
October, 1999
96F (IIIR ch.)
Tested
6E (Ich.);
September, 2000
96F (IIIR ch.)
ch – means chromosoma
4
9 (new)
All sites where
Penelope insertions
were found
21C, 6E, 54F, 100F
22F, 70D, 100B,
55A, 54A, 28E, 95E,
56C, 92D
(13 total)
_
5
17F, 24A, 51B, 55F,
57B (5 total)
Drosophila melanogaster
transformation with the Penelope
transposable element
Sterile
ovaries
Agametic
ovarioles
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
3
1
2
hsp70Aa
4
4
hsp70Ab
2
1
54
7
5
CG18347
CG3281 aur
3
55
5
6
7
EPgy2
5
5
CG12213
53
P-element construct
hsp70Aa
hsp70Ab
∆2-3
EPgy2
♀
Yw
Yw
P(y+w+)
yw
Df yw
yw
♀ yw
+
+
Df
♂
X
+
+
♀
∆2-3
P(y+w+)
X
yw
♂
X
yw
P(y+w+)
∆2-3
P(y+w+)
♂
+
+
+
P(y w ) P(y+w+) n
yw
X
+
Selection of males with
enhanced eye color
yw P(y+w+) P(y+w+) n
yw P(y+w+) P(y+w+)
♀
X ♂
yw
+
+
Selection of strains, containing additional copies
of construction
n
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
57%
Strains from US-2
hsp70Aa
hsp70Ab
43%
-229(1) 200(1)
-135(4)
-97(3)
+1
H
H
g
-160(1)
g
g
H g
-96(3)
H g
-40(1)
T
87%
Strains from US-4
hsp70Aa
hsp70Ab
13%
CGGCGCAC target duplication
-240(1)
-174(1)
-137(1)
-135(4)
-97(12)
-42(2) -28(1)
+1
H
H
g
g
-144(1)
g
H g
-96(5)
H g
T
-40(1)
GGCGCACT target duplication
Сравнение экспериментальной и естественной транспозиции

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.

