MOLECULAR AND CELLULAR BIOLOGY, June 1991, p. 3395-3398
Vol. 11, No. 6
0270-7306/91/063395-04$02.00/0 Copyright © 1991, American Society for Microbiology
Cloning by Differential Screening of a Xenopus cDNA That Encodes a Kinesin-Related Protein RENE LE GUELLEC, JEANNIE PARIS,t ANNE COUTURIER, CHRISTIAN ROGHI, AND MICHEL PHILIPPE* Laboratoire de Biologie et Genetique du Developpement, Centre National de la Recherche Scientifique, URA 256, Campus de Beaulieu, Universite de Rennes 1, 35042 Rennes Cedex, France Received 17 December 1990/Accepted 26 March 1991
By differential screening of a Xenopus egg cDNA library, we selected nine clones (Egl to Eg9) corresponding are deadenylated and released from polysomes soon after fertilization. The sequence of one of these clones (Eg5) revealed that the corresponding protein has the characteristic features of a kinesin-related protein. More specifically, Eg5 was found to be nearly 30% identical to a kinesin-related protein encoded by bimc, a gene involved in nuclear division in Aspergillus nidulans. to mRNAs which
For most animals, the developmental period following fertilization is characterized by a state of very rapid cellular proliferation (6). In the case of xenopus, the first cleavage takes place 1.5 h after fertilization and is followed by 11 almost synchronous cell divisions which occur every 30 min (23). The onset of transcription is clearly detected only after cleavage 12 at a stage called the midblastula transition (23). In the presence of dactinomycin, embryos develop up to the midblastula transition, whereas this development is blocked by puromycin or cycloheximide (3, 20). Similar results have been obtained for maturation, which is independent of new transcription but requires de novo translation (15). Qualitative analysis of the proteins synthesized in oocytes (stage VI), in unfertilized eggs, and in embryos has shown that during maturation and after fertilization, new proteins appear but others are no longer synthesized (5, 19, 29). This suggests that the synthesis of specific gene products necessary for maturation, for the metaphase block in unfertilized eggs, and then for rapid proliferation is regulated at the translational level from the bulk of maternal mRNA. By differential screening of a cDNA library prepared with poly(A)+ RNA from unfertilized eggs, we isolated 11 nonoverlapping sequences which undergo either adenylation or deadenylation after fertilization (26). Nine of them which constitute the Eg family correspond to RNAs which are specifically deadenylated (24-26) and released from polysomes after fertilization (24, 26). In a previous work, we have shown that Egl has very high homology to p34cdc2 but is clearly functionally different from the kinase subunit of the maturation-promoting factor (24). In the present report, we describe the characteristics of EgS, which clearly belongs to the kinesine-related protein family. Kinesin was first discovered during studies of organelle transport in giant squid axons (1, 30). Subsequently, kinesin has been found in a variety of organisms and cell types, including avian and mammalian neuronal tissues (4, 30), sea urchin eggs (28), cultured cells (22), and Drosophila melanogaster (27). Biochemical studies of kinesin protein revealed that it is a tetramer consisting of two heavy and two light chains (2, 14). The gene that codes for the drosophila heavy chain has been cloned and characterized (33, 34). *
More recently, genes that encode kinesin-related proteins were identified by genetic criteria in D. melanogaster (7, 35) or by molecular genetic critera in Saccharomyces cerevisiae (18), Aspergillus nidulans (8), D. melanogaster (16), and Schizosaccharomyces pombe (11). The KAR3 mutant of S. cerevisiae is defective in karyogamy (18). Mutations in the bimc gene of Aspergillus nidulans prevent spindle pole body separation and nuclear division (8). In S. pombe, cut7+ is referred as a gene involved in spindle formation (11). The product of the drosophila claret gene is clearly implicated in chromosome segregation and is active in meiosis (7). The nod gene is required for distributive segregation of nonexchange chromosomes during meiosis in D. melanogaster (35). The sequencing strategy, the nucleotide sequence, and the predicted amino acid sequence of Eg5 are shown in Fig. 1A and B. The cloned EgS cDNA is 3,651 nucleotides long and contains 193 nucleotides of 5'-flanking sequence, an open reading frame of 3,180 nucleotides, and a 3'-untranslated region of 278 nucleotides which contains a potential polyadenylation signal (AAUAAA) at nucleotide position 3614 to 3619. The open reading frame codes for a predicted polypeptide 1,060 amino acids long with a relative molecular mass of 119,332 Da. The secondary structure predicted (10, 32) for EgS protein is shown in Fig. 1C. The predicted EgS protein has two globular domains separated by an a-helical region, which is a characteristic of all known kinesin and kinesinrelated proteins. Like all of these proteins, EgS also possesses a putative motor domain, which is shown in Fig. 2 (from amino acids 1 to 358). Figure 2 also shows the similarities of the motor domain encoded by EgS (defined as amino acids 1 to 358, corresponding to the end of Kar3) to the proteins encoded by bimc (8), the drosophila kinesin heavy chain gene (34), cut7+ (11), claret (7), and KAR3 (18), which are, respectively, 53.3, 49.7, 38.9, 29.9, and 28.6%. At amino acid positions 92 to 107, Eg5 possesses a putative ATP-binding site (IFAYGQTXXGKTXTM) which is conserved in all five sequences. As in the drosophila and squid kinesin heavy chains (13, 34) and the nod (35)-, bimc (8)-, and cut7+ (11)-encoded proteins, the proposed motor domain of Eg5 is located at the amino terminus. In contrast, in the KAR3 (17)- and claret (7, 33)-encoded proteins the mechanochemical domain is situated in the carboxy-terminal region. The sizes of the proteins encoded by EgS, bimc, cut7+, and the drosophila heavychain gene are in the same range, at 1,060, 1,211, 1,073, and
Corresponding author.
t Present address: Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545. 3395
3396
NOTES
A
MOL. CELL. BIOL.
E
_
i_ 0
*6
o
0
*
>
~ 500 bp
B
gottggctgtggctgtcggaagcgcgattttcaaatgtgtctgctgcgaggag3agccgt 60
gcttgcggaacgattttaacgcttggctgtagg4agtgggatcttcgcgctctggttact
120
tcaactgtctctgctcggggaagggaatcgtgactgcttattggagataactatgtcttc 180 ccaaaattcttttATGGCCAGCAAGAAGGAGGACAAAGGGAAAAACATCCAGGTTGTGGT 240 M
A
S
K
K
E
D
K
G
K
N
I
0
V
V
v16
CCGATGCCGGCCCTTCAATCAGTTGGAACGCAAGGCGAGCTCTCACTCTGTGTTGGAATG 300 N
C
R
P
F
N
Q
L
E
R
K
A
S
S
S
H
V
L
E
C 36
TGATTCTCAGAGGAAAGAAGTCTATGTTCGGACCGGAGAAGTAAACGACAAACTGGGAAA 360 0
S
D
R
K
E
V
Y
V
R
G
T
E
V
D
N
K
L
G
K
SN
GAAAACCTATACGTTTGACATGGTGTTTGGGCCTGCAGCCAAGCAAATTGAGGTGTACAG T
K
Y
T
F
0
N
V
F
G
P
L
A
G
A
A
K
0
R
I
E
V
Y
420 R 7N
0
1 136
E
L
I S56 720 0176
I
S196
AAGTGTTGTGTGCCCCATCTTGGATGAAGTTATTATOGGATATAACTGCACAATCTTTGC 480 S V V C P I L D E V I N G Y N C T I F A 96 GTATGGGCAGACTGGCACAGGCAAGACCTTTACTATGGAAGGGGAAAGATCATCAGATGA 540 Y K 0 T G T G K T F T M E G E R S S 0 EK 16 AGAGTTTACCTGGGAACAGGACCCTCTGGCAGGAATTATTCCTCGAACGCTGCATCAGAT 600 F
E
T
0
I
P
L
H
L
S
E
N
G
T
E
F
S
v
K
V
S
1
1
E
L
F
D
L
L
S
P
S
P
D
V
G
E
W
E
D
P
I
T
ATTTGAGAAGCTCTCTGAGAACGGCACAGAGTTTTCGGTGAAAGTCTCTCTCCTGGAAAT 660 F
E
6
N
E
CTACAACGAAGAACTCTTTGATCTGTTAAGCCCATCTCCCGATGTTTGGGAAAGGCTGCA Y
6
AATGTTCGATGATCCTCGGAACAAAAGAGGAGTCATTATCAAAGGGCTGGAGGAGATATC 780 F
H
0
D
P
R
N
K
R
G
V
I
I
K
G
L
E
E
TGTGCATAACAAGGATAGGWTGTACCATATATTGGAGAGAGGTGCAGCCAGGAGGAAAAC 840
V 6 N K D E V Y N I L E R G A A R R K T 216 TGCTTCTACTCTGATGAATGCATATTCCAGTCGATCCCATTCCGTGTTCTCGGTCACCAT 900 A S T L 6 N A Y S S R S N S V F S V T I 236
TCACATGAAAGAAACTACTGTTGATGGAGAAGAGCTTGTGAAGATCGGGAAGCTGAACTT 960 H
K
M
E T
E
V
L
V
K
I
G
K
L
N
L 256
GGTGGATCTGGCGGGAAGTGAAAATATTGGTCGTTCTGGAGCTGTGGACAAAAGAGCACG V D L A G S E N I G R S G A V 0 K R A R CGAGGCTGGAAATATCAACCAATCCCTGCTCACCTTGGGCAGGGTAATAACTGCTCTGGT E A G N I N 0 L L T L G R V I T7 A V GGAGAGAACTCCTCATATTCCATACAGAGAATCCAAACTCACAAGGATCCTGCAGGATTC E R T P H I P Y R E S K L T R I L 0 D S TCTTGGAGGCAGGACAAAAACCTCTATCATTGCAACAGTGTCACCTGCATCCATTAATCT L
G
G
R
T
K
T
S
I
I
A
T
V
S
P
A
S
I
N
N
K
L
GGAGGAAACTGTGAGCACTTTGOACTATGCCAACAGAGCAAAGAGTATTATGAATAAGCC E
T
E
V
S
T
L
0
Y
A
R
N
A
K
I
S
M
P
AGAGGTTAACCAGAAACTTACAAAGAAGGCACTCATCAAGGAGTACACCGAAGAGATTGA E
V
N
0
K
L
T
6
K
A
I
L
K
E
T
Y
E
1020 276
1080 296 1140 316 1200 336 1260 356 1320
E
I
E 376
V
GCGACTTAAGAGGGAACTTGCTGCAGCCAGAGAGAAGAATGGAGTTTATTTATCCAGTGA 1380 R L K R E L A A A R E K N G V Y L S S E 395 GAACTATGAACAATTACAGGGAAAAGTTCTGTCTCAAGAGGAGATGATTACGGAGTACAC 1440 N Y E 0 L 0 G K V L S 0 E E M I T E Y T 416 TGAGAAAATAACTGCCATGGAGGAGGAACTGAAAAGCATCAGTGAGCTGTTTGCTGACAA 1500 E K I T A M E E E L K S I S E L F A D N436 TAAGAAGGAGCTGGAGGAGTGCACCACCATCCTTCAGTGCAAGGAGAAGGAACTGGAGGA 1560 K K E L E E C T T I L 0 C K E K E L E E 456 AACACAGAACCACCTGCAAGAATCAAAGGAACAGTTGGCTCAGGAATCATTTGTGTGTC 1020 T 0 N N L 0 E S K E 0 L A 0 E S F V V S 476 TGCCTTTGAGACTACAGAAAAGAAGCTTCACGGCACAGCAAACAAGTTATTGAGCACAGT 1680 A F E T T E K K L N G T A N K L L S T V A96 GAGGGAGACAACAAGAGATGTGTCGGGTCTCCATGAGAAATTGGACAGAAAAAAAGCTGT 1740 R
E
T
T
R
D
V
S
G
L
H
E
K
L
D
R
K
K
A
0
0
H
N
F
0
V
N
E
N
F
A
E
0
M
D
R
R
F
0
R
V
D
0
0
G
6
L
D
F
AGACCAGCACAACTTTCAGGSTCATGAGAACTTTGCGGAACAAATGGATAGACGCTTCAG
SIG
1800 S 536
TGTGATTCAGCGAACTGTGGATGATTACAGTGTGAAGCAGCAAGGCATGCTGGATTTCTA 1660 I
V
T
0
Y
S
V
K
Y SSG
TACAAACTCCATCGACGACCTTCTGGGCGCCAGTTCCTCTCGACTGAGTGCAACTGCCTC T
N
S
I
0
D
L
L
G
A
S
S
S
A
L
S
A
T
A
1920 S 576
TGCTGTTGCCAAGTCCTTTGCCTCGGTGCAGGAAACTGTAACAAAGCAAGTGTCGCACAG 1980 V
A
A
K
S
F
A
S
V
0
E
T
V
T
0
K
V
S
H
S 596
TGTTGAGGAGATACTGAAGCAGGAAACCTTGTCTTCCCAAGCTAAAGGTGATCTGCAGCA 2040 V E I L K 0 E T L S S 0 A K G D L 0 0616
GCTTATGGCTGCGCATCGCACAGGACTGGAAGAGGCACTAAGGTCCGATTTGCTTCCAGT 2100 L
A
A4
H
R
T
G
L
E
E6
L
R
D
S
L
L
P
V636
TGTGACTGCTGTTTTGGACCTAAATTCTCATTTGAGTCATTGCTTGCAGAACTTCCTAAT 2160 V
N
0 N F L I 656 TGTGGCCGACAAGATTGATTCTCATAAAGAAGATATGAATTCCTTCTTCACCGAACATTC 2220 V A 0 K I 0 5 H K E 0 M N S F F T E H S 676 TAGATCGTTGCACAAACTTCGGCTGGATTCTAGTTCTGCCTTGTCCAGTATTCAGTCAGA 2280 R S L 0 K L R L 0 S S S A L S S I 0 S E 696 ATATGAAAGCCTAAAAGAGGACATTGCSACAGCTCAGTCCATGCATTCAGAGGGGGTGAA 2340 Y E S L K E 0 I A T A 0 S M H S E G V N 716 TAATCTTATCAGTTCGCTACAAAACCAGCTGAACTTGCTTGGCATGGAGACCCAACAACA 2400 N L I S S L 0 N 0 L N L L G M E T 0 0 0 736 ATTTTCTGGATTTCTGTCCAAAGGAGGAAAGCTGCAGAAGTCTGTGGGGAGTCTGCAGCA 2460 F S G F L S K G G K L 0 K S V G S L 0 0 756 AGATCTGGACTTGGTTTCCTCGGAAGCCATAGAATGTATCTCTTCCCATCACAAOGAAGCT 2520 D L D L V S S E A I E C I S S 0 14 K K L 776 T
V
A
L
D
L
N
S
L
S
H
C
L
CGCTGAGCAGTCTCAGGACGTGGCTGTGGAGATCAGGCAGCTGGCAGGCTCTAATATGAG 2580 0
E
A
S
0
0
V
L
V
E
I
R
0
L
A
G
S
N
M5 796 2640 I S 61
CACCTTGGAGGAGTCCAGCAAACAGTGTGAGAAGCTCACCAGCAGTATCAATACCATCTC T
E
L
E
5
S
K
0
C
E
K
L
T
S
I
S
N
T
TCAGGAAAGCCAGCAGTGGTGTGAAAGTGCCGGCCAGAAGATTGACTCTGTTTTAGAGGA 2700 Q E S 0 0 W C E S A G 0 K I 0 S V L K E 636
GCAAGTGTGTTACCTGCATTCCAGCAAAAAGCATCTCCAGAACTTACACAAGGGTGTAGA 2760
0 V C Y L H S S K K H L 0 N L H K G V E 656 AGATAGCTGTGGATCATCGGTOGTAGAAATTACCGACCGTGTGAACGCACAGCGCCAGGC 2620 D S C G S S V V E I T D R V N A 0 R 0 A 876 TGAGGAGAAGGCATTAACCAGTTTGGTGGAA TCAGGGATGACCAGGAGATGGTTGG 2880 E
E
K
A
L
T
S
L
V
E
0
V
0
V
R
0
0
D
E
M
v
H
S
G
196
AGAACAGCGCtTGGAACTCCAAGAACAAGTACAGAGCGGCCTGAACAAAGTACATAGCTA 2940 0
E
R
L
E
L
0
E
0
S
G
L
K
N
V
Y 916
CCTCAAGGAGAACTCAGGAATGATGTCCCAACAGGTACAACGCCCCAGAGGAGGGATTA 3000 L K E E L R N 0 V P T G T T P 0 R R 0 Y936
CGCGTACCCATCTTTGCTTGTTAAAACAAAACCCAGGGATGTGCTGTTGGAGCAGTTTAG A Y P S L L V K T K P R D V L L E 0 F R GCAACAGCAGCAAGAATATCTGGAATCCATATCCAGCGTGATCTCTGAGGCTGTGGAACC 0 0 0 0 E Y L E S I S S V I S E A V E P GCCAGTGGAGCAGGATTCTCTAGAAGATGAGCCACCGGTTGCAGTTAATGACAGCGTCAT P V E 0 D S L E D E P P V A V N D S V 1 CAGTGAGAGATCCTGCATTGATCTCAGTATGACTTGTCAGGAGAAAGGAGGAATTCGATT S E R S C I D L S H T C 0 E K G G I R F CTTCCAGCAAAAGAAAGCACTTCGGAAGGAAAAGGAAAACCGGGGAAATACAACACTTTT F 0 0 K K A L R K E K E N R G N T T L L GGAGAGATCTAAAATCATGGACGAGGTGGACCAAGCTCTTACGAAATCTAAACTTCCCCT E R 5 K I H 0 E V 0 0 A L T K S K L P L GCGAATGCAGAACtga agcacttgcttaacccScatgatgatgtgcggctttgtctOtct 6 0
R
N
3060
956 3120 976
3180
996 3240 1016 3300 1036 3360
05S
3420 1060
gtgcagaaa,lttga.tacatttttaactgtttstgStatccgggtgttgcgtatgSagaa
3460
aattttattcatcaataaatattcccttttctataaaaaoaaaaaaaaaa
3651
ettttacttatcttggttatatatatgtgtgtctatcgtcacttgtatgtcaaatattaa 3540 aaatactttctaaagtagaacttctcaggctttacgagttagttatggaaaagtStattt 3600
c
II§ 1 1 I J LW IJ IfI
GOR TURNS GO ALPHA
GOR
HELICES
i1 IL I I lil 1V]FIJI JII LLJ1F J1 Tl L
BETASHEETS
I
0
500
975 amino acids, respectively. There is no obvious similarity between the amino acid sequences of Eg5 and the drosophila heavy chain in the carboxy-terminal regions of the molecules. By contrast, as shown in Fig. 2B, significant similarity was found among the proteins encoded by Eg5, cut7+, and bimc. In particular, from amino acids 911 to 956, more than 35% of the Eg5- and bimc-encoded amino acids are identical and more than 50% are of the same family. The similarity extends beyond sequence comparisons; in particular, the structures of the central regions of the proteins encoded by Eg5, bimc and cut7+ are very closely related, which could suggest similar functions for these gene products. In a previous work (26), we have shown that Eg5 RNA translation is regulated during very early development. In eggs, the RNA is found in both the poly(A)+ fraction and polysomes. After fertilization, Eg5 RNA is deadenylated and concomitantly released from the polysomes. To know when Eg5 RNA appears and when it is adenylated during oogenesis, we performed Northern (RNA) analysis of total RNAs extracted from oocytes (stages I to VI), eggs, and premidblastula transition embryos by using a 32P-labeled Eg5 cDNA probe. Considering that a fixed quantity of total RNA (10 Rg) was used and that rRNA constitutes a progressively larger proportion of the total RNA during oogenesis (i.e., 25% in stage I oocytes versus 90% in stage VI oocytes), the decrease in the autoradiogram signals observed in Fig. 3A reflects the dilution of these transcripts in the pool of total RNA. Taking this into account, quantitation of autoradiograms from these Northern analyses confirmed that the number of these transcripts per oocyte or embryo was approximately constant from stage III of oogenesis to the midblastula transition (Fig. 3B). Closer examination of the data (Fig. 3A) shows that Eg5 transcripts appear to be larger in eggs than in either oocytes or embryos, suggesting that the RNA is adenylated only in eggs. When poly(A)+ and poly(A)- RNAs were separated from the total RNAs extracted from oocytes at different stages and analyzed by Northern blotting with Eg5 as the probe, we never found Eg5 in the poly(A)+ fraction (Fig. 3C). Moreover, in its 3'untranslated region (at nucleotide positions 3430 to 3436), Eg5 RNA possesses a sequence motif [UUUU(A)AU] similar to those shown to be necessary for maturation-specific adenylation of Xenopus mRNA (9, 17). This supports the hypothesis that during maturation Eg5 translation is regulated through adenylation, as recently reviewed (12, 31), and also suggests that the protein is present in eggs but not in oocytes. Antibodies against bovine kinesin reveal a pair of 120-kDa polypeptides in a crude Xenopus egg extract (28). After fertilization, Eg5 RNA is no longer detected in polysomes, suggesting that its further translation is not necessary for early development. This is in agreement with the notion that the 12 rapid cell divisions which follow fertilization
1000
FIG. 1. Nucleotide sequence of Egl cDNA and predicted amino acid sequence of the protein. (A) Restriction map and sequencing strategy. The arrows denote the extent and direction of sequence reading of each fragment. (B) Nucleotide and amino acid sequences. The nuclear polyadenylation signal is indicated by solid underlining (nucleotide positions 3614 to 3619). The putative cytoplasmic polyadenylation sequence specific for maturation is indicated by broken underlining (nucleotide positions 3430 to 3436). (C) Secondary structure prediction for Eg5 protein. The secondary structure of Eg5 protein predicted by the method of Garnier et al. (10) is shown. Regions predicted to be a turns (GOR turns), a helices (GOR alpha helices), or ,B sheets (GOR beta sheets) are indicated by the elevated segments.
VOL. 11, 1991 Eg5
.......
BINC
.
CUT7+
6KKKG
?
... (SO)DHALNDENE
L1INTVjLR
.(375)TLHNELQELR .... YTF
Eg5
BISC
IS
'AC '
E~VVLQTE....G%
IV5ETGEVN 51 KLSHGP 51
TSGAMGA LAI;SDPSSN P VAlF FP.... .1LQSID NR CTTY.. .DES;N4jEENCIS
ft
LE
53
T1FAYGQTGTG
107
YNCTIPAYOQTOTO Y IFAY PAY
107 100 106
LFEN
.
~
~
~
IIQNTAQVHEF
KAR3
Egs szwc CUT7+ DEINESIN
N
P..b D
IINC
CUT7+ DKINESIN CLARET
KAR3
LQNF&Ft
Y
Eg5
NDO
215 210 211 197 203
............................
.:tDPNHL4 .NVTSCKLESEENVRIILKEANK
S ......... QENLENQRELS.
NIGPSGA
RINV
QVBD
T_TSIIATVSPA P
SLOOGR 315 IPYRESELT 328 DS IPYRESKLT 300 IY KLELG 301 . .EQ I KL I E 1 317 PDST I _ 380 LSLE 375 I 37TLDY5TN LE
TKE IA 388 LV TL0 PA AKI3VIC KRDC ENTKAERNRYLN NSVAN SNNSONF 361 NFl I NVKy 355 FVN BLYSRK NGVY
.*ESICL435 RIISTS RNLEN A DLLC EVLDLNVKSSRE YV9SN 448
LKGKV(609)
365 363
EN.4
KE A>WTT NDTLAA TND
LQLX NSs
Q
L
*mG2Ktl DK
IMLR VSENEK &E EKRKK
493
TTvRrB
495 508 553
wRAS STEV SDVTKR;D~~~~~~~~~8~8AFQTRNAK E 555 ELQVHNFAEQMDRtRFS%1;QR DQ^ FTRK L 555 aimc L3V3gN I DE4 HC B~~~~~~I QY KEYIA EERNNE NNFI.KFNLLTHLRSFHGSFTDETNGYFTL4J 568
E
'S
DYSVKQQG
D
...ASVQETVTKQV* S#EILKQETLSSQA
DFYTI&DA ATSAVA ETTSVKVNFIATE IERTIOLSEYNR.DAACNNAAE A NNNVLEEIEDL 615 i4TI ISNTKITEHFCHF DEALQSARSSCAVPNSSLDLIVSELKDS 628
610
CUT7?
NDFNA&EE
EgS
NCLQ4IVADUIr B.. ."NLGKDL4; FCT 672 KNSEDALEHSLQDISNSSQEL0NGIGSSELI E :DES. .. YRQLVQELYNLQHT 685 FFTR DSRSL4.DSS1 IQSETES3 mAmNN ... LISSLQLN 727 W8QNIETTI SH IE91#%AEARREXLNSQIEA 730 HLSENEI NlEL R.
BINC CUT?7. Eg5 BINC
CUT7+ Bg5
"INC CUT7+
EgS
8INC CUT
+
EgS BINC
CUT7+ Eg 5
BIMC
CUT7.
Eg8S
BIMC CUT7+ Eg5 BINC BIMC
8
-
;:f
*
*
*
30 20
.
-
1010-
1
.
f-
11
0
V
IV
III
,.,
rt
SOt
-
+
4
UFE
VI
6
8
10
Lhc
c
KGD&QLMNAAHRFUE4RSDLLRVTAVL REEVKSKVGEG AkAI8ARISEEI GEOK
S 670
HEESQEELNYGMEDIDA. . LVKTCTTSLND'I LSDYISDQKSKFESKQQDLIASGK ON ISSHREQSQDVL DLDLVSS GPLSW . .GGKLQKS EVFKSBQ DVNASB RL WEES DGVRTEISAmLEQATTQHD I RANEINNNR FLRNAAS IVGANK ESLD ILHSHLNDT IVSNF
790 803
I NAGSNMS&ESC TISQESQQWCESAGQKIDSVE#YL1# rHETVRIVDVQVDDNGRQNEA DE lrKLQNDWEAFDQRNSTI PDFJKA"P
850
E*fJKTVENGSQLOS 19
. KKHLQN GV C NGRYRD AT IA SEDNTKEEQLL L
IHNSNS
T
-+
II1 + IV
T
V
-+
+
VI
T
-
UFE
743
784 844
FIG. 3. (A) Northern blot analysis of total RNAs extracted from eggs (UFE), and embryos 4, 6, 8, and 10 h after fertilization. Samples of RNA (10 ,ug) were separated on agarose gel containing 6% formaldehyde and blotted onto nylon membrane (Hybond [Amersham]). Purified inserts were 32P labeled with random primers to a specific activity of 5 x 108 cpm ,Lg-1. Hybridization was carried out in 50% formamide-1% sodium dodecyl sulfate-10 x Denhardt's solution-10% dextran sulfate-1% PPi-1 M NaCl-0.05 M Tris-HCl (pH 8) at 42°C overnight. Filters were extensively washed in 2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.5% sodium dodecyl sulfate at 65°C. (B) Quantitative analysis of Eg5 RNA during oogenesis and very early embryogenesis. MBT, midblastula transition. (C) Total RNAs were extracted from oocytes (stages I and II, III and IV, and V and VI) and unfertilized eggs (UFE). Poly(A)+ was separated from poly(A)by oligo(dT) chromatography. Samples [10 pLg of total RNA, 10 ,ug of poly(A)- RNA, and 200 ng of poly(A)+ RNA] were analyzed by Northern blotting as described for panel A.
oocytes (stages I to VI), unfertilized
DHCLALAESQKQGVNLEVQ*JRLLQKVKEH 863
EITDRVNAQRQAEEEALTSLVEC6DDQEMVGORL4s DSYSSIEGRVEHLTGRNNQFQQEATNHHATLEIIA; 909 903
NNDNLIDSIETPHTELQK.ITDIeLKGTTSLANHTNFU 922 NRF1;*?FCPR*MLEQ. QQQ1 961
QS.VH*MLRE NTREE NRSMEYWG DLS S EL N"YSK} VPFS:SH*SR KETT ASWTRDSSLI GLGDE ETTIEDT E
+ 11
433
EENI
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NOTES
...
4SIV1SEAVEPPVEQDS11EPPV
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SERSCI DI.1TCQE9GI RFFQQKK 1021 1029 1024
THAPTVTNVNPSNTGLREVDANVAA VLFNPDQLSGPSSSPGGSSKGFVYN LDPKKFVRETYTSSNQTNWDVYDKPSNSSRTSLLRSSRl5YSKE1
ALRKEKENRGIf;LLERSEIMDEVDQALTKSELf.RNQN RPLVYSTGEKSE QDGSPVVSPDSATEAEGCNNU'SKRRRSNSVVADTKLPNE.LARRNA
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1089
FIG. 2. The predicted amino acid sequence of the motor domain of Eg5 is aligned with those encoded by Aspergillus bimc, S. pombe cut7+, the drosophila kinesin heavy-chain gene, saccharomyces KAR3, and drosophila claret. The numbers of the residues at which the bimc, cut7+, claret, and KAR3-encoded proteins extend in the amino-terminal direction and the drosophila heavy chain extends in the carboxy-terminal direction are in parentheses. Alignment of the carboxy-terminal parts of the Eg5-, bimc- and cut7+-encoded molecules is also shown. Identical residues are boxed. The central regions of the Eg5-encoded protein (from amino acids 366 to 535) and the bimc-encoded protein (and also the cut7+-encoded protein, to a lesser extent) show conserved amino acids (dots) spaced by six amino acids. Most of these conserved amino acids are leucines.
could only be under the translation control of cyclins, as suggested by in vitro experiments using egg extracts (21). Moreover, the fact that Eg5 RNA was not detected in adult tissues (26) suggests that it is involved in cell proliferation. Taking these points into account, and also the similarity among EgS, cut7+, and bimc, one can postulate that Eg5encoded protein is involved in nuclear division and may be the counterpart of bimc-encoded protein in xenopus. Nucleotide sequence accession number. The sequence described here has been assigned EMBL accession no. X 54002. We are grateful to Michel Kress and H. B. Osborne for very helpful discussions, to Chris Ford and Sharyn Endow for critical reading of the manuscript, and to F. de Sallier Dupin for preparing the manuscript. This work was supported by the Association pour la Recherche sur le Cancer, the Ministere de la Recherche et de la Technologie, la Ligue Departementale d'Ille et Vilaine de Recherche contre le Cancer, and la Fondation pour la Recherche Medicale.
3398
NOTES
REFERENCES 1. Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson. 1985. Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport. J. Cell Biol. 100:1736. 2. Bloom, G. S., M. C. Wagner, K. K. Pfister, and S. T. Brady. 1988. Native structure and physical properties of bovine brain kinesin and identification of the ATP binding subunit polypeptide. Biochemistry 27:3409-3416. 3. Brachet, J., H. Denis, and F. Devitry. 1964. The effects of actinomycin D and puromycin on morphogenesis in amphibian eggs and Acetabularia mediterranea. Dev. Biol. 9:398-434. 4. Brady, S. T. 1985. A novel brain ATPase with properties expected for the fast axonal transport motor. Nature (London) 317:73-75. 5. Bravo, R., and J. Knowland. 1979. Classes of proteins synthesized in oocytes, eggs, embryos and differentiated tissues of Xenopus laevis. Differentiation 13:101-108. 6. Davidson, E. H. 1986. Gene activity in early development, 3rd ed. Academic Press, Inc., Orlando, Fla. 7. Endow, S. A., S. Henikoff, and L. Soler-Niedziela. 1990. Mediation of meiotic and early mitotic chromosome segregation in Drosophila by a protein related to kinesin. Nature (London)
345:81-83. 8. Enos, A. P., and N. R. Morris. 1990. Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans. Cell 60:1019-1027. 9. Fox, C. A., M. D. Sheets, and M. P. Wickens. 1989. Poly(A) addition during maturation of frog oocytes: distinct nuclear and cytoplasmic activities and regulation by the sequence UUUUUAU. Genes Dev. 3:2151-2162. 10. Garnier, J., D. J. Osguthorpe, and B. Robson. 1978. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120:97-120. 11. Hagan, I., and M. Yanagida. 1990. Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene. Nature (London) 347:563-566. 12. Jackson, R. J., and N. Standart. 1990. Do the poly(A) tail and 3' untranslated region control mRNA translation? Cell 62:15-24. 13. Kosika, K. S., L. D. Orecchio, B. Schnapp, H. Inouye, and R. L. Neve. 1990. The primary structure and analysis of the squid kinesin heavy chain. J. Biol. Chem. 265:3278-3283. 14. Kuznetsov, S. A., E. A. Vaisberg, N. A. Shanina, N. N. Magretova, V. Y. Chernyak, and V. I. Gelfand. 1988. The quaternary structure of bovine brain kinesin. EMBO J. 7:353-356. 15. Maller, J. L. 1985. Regulation of amphibian oocyte maturation. Cell Differ. 16:211-221. 16. McDonald, H. B., and L. S. B. Goldstein. 1990. Identification and characterization of a gene encoding a kinesin-like protein in Drosophila. Cell 61:991-1000. 17. McGrew, L. L., E. Dworkin-Rastl, M. B. Dworkin, and J. D. Richter. 1990. Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element. Genes Dev. 3:803-815. 18. Meluh, P. B., and M. D. Rose. 1990. KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell 60:1029-1041.
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19. Meuler, D. C., and G. M. Malacinski. 1984. Molecular aspects of early development. Plenum Publishing Corp., New York. 20. Miake-Lye, R., J. W. Newport, and M. Kirschner. 1983. Maturation promoting factor induces nuclear envelope breakdown in cycloheximide arrested embryos of Xenopus laevis. J. Cell Biol. 97:81-91. 21. Murray, R. W., and M. W. Kirschner. 1990. Dominoes and clocks: the union of two views of the cell cycle. Science 246:614-621. 22. Neighbors, B. W., R. C. Williams, Jr., and J. R. McIntosh. 1988. Localization of kinesin in cultured cells. J. Cell Biol. 106:11931204. 23. Newport, J., and M. Kirschner. 1982. A major developmental transition in early Xenopus embryos. I. Characterization and timing of cellular changes at the midblastula stage. Cell 30:675686. 24. Paris, J., R. Le Guellec, A. Couturier, K. Le Guellec, F. Omilli, J. Camonis, S. MacNeill, and M. Philippe. 1990. Cloning by differential screening of a Xenopus cDNA coding for a protein highly homologous to cdc2. Proc. Nati. Acad. Sci. USA 88: 1039-1043. 25. Paris, J., H. B. Osborne, A. Couturier, R. Le GuelHec, and M. Philippe. 1988. Changes in the polyadenylation of specific stable RNA during the early development of Xenopus laevis. Gene 72:169-176. 26. Paris, J., and M. Philippe. 1990. Poly(A) metabolism and polysomal recruitment of maternal mRNAs during early Xenopus development. Dev. Biol. 140:221-224. 27. Saxton, N. M., M. E. Porter, S. A. Cohn, J. M. Scholey, E. C. Raff, and J. R. McIntosh. 1988. Drosophila kinesin: characterization of microtubule motility and ATPase. Proc. Natl. Acad. Sci. USA 85:1109-1113. 28. Scholey, J. M., M. E. Porter, P. M. Grissom, and J. R. McIntosh. 1985. Identification of kinesin in sea urchin eggs and evidence for its localization in the mitotic spindle. Nature (London) 318:483-486. 29. Smith, R. C. 1986. Protein synthesis and messenger RNA levels along the animal-vegetal axis during early Xenopus development. J. Embryol. Exp. Morphol. 95:15-35. 30. Vale, R. D., T. S. Reese, and M. P. Sheetz. 1985. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42:39-50. 31. Wickens, M. 1990. In the beginning is the end: regulation of poly(A) addition and removal during early development. Trends Biol. Sci. 15:320-324. 32. Wolf, H., S. Modrow, M. Motz, B. A. Jameson, G. Hermann, and B. Foertsch. 1988. An integrated family of amino acid sequence analysis programs. Comput. Appl. Biosci. 4:187-192. 33. Yang, J. T., R. A. Laymon, and L. S. B. Goldstein. 1989. A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses. Cell 56:879-889. 34. Yang, J. T., W. M. Saxton, and L. S. B. Goldstein. 1988. Isolation and characterization of the gene encoding the heavy chain of Drosophila kinesin. Proc. Natl. Acad. Sci. USA 85:1864-1868. 35. Zhang, P., B. A. Knowles, L. S. B. Goldstein, and R. S. Hawley. 1990. A kinesin-like protein required for distributive chromosome segregation in Drosophila. Cell 62:1053-1062.
Vol. 11, No. 6
0270-7306/91/063395-04$02.00/0 Copyright © 1991, American Society for Microbiology
Cloning by Differential Screening of a Xenopus cDNA That Encodes a Kinesin-Related Protein RENE LE GUELLEC, JEANNIE PARIS,t ANNE COUTURIER, CHRISTIAN ROGHI, AND MICHEL PHILIPPE* Laboratoire de Biologie et Genetique du Developpement, Centre National de la Recherche Scientifique, URA 256, Campus de Beaulieu, Universite de Rennes 1, 35042 Rennes Cedex, France Received 17 December 1990/Accepted 26 March 1991
By differential screening of a Xenopus egg cDNA library, we selected nine clones (Egl to Eg9) corresponding are deadenylated and released from polysomes soon after fertilization. The sequence of one of these clones (Eg5) revealed that the corresponding protein has the characteristic features of a kinesin-related protein. More specifically, Eg5 was found to be nearly 30% identical to a kinesin-related protein encoded by bimc, a gene involved in nuclear division in Aspergillus nidulans. to mRNAs which
For most animals, the developmental period following fertilization is characterized by a state of very rapid cellular proliferation (6). In the case of xenopus, the first cleavage takes place 1.5 h after fertilization and is followed by 11 almost synchronous cell divisions which occur every 30 min (23). The onset of transcription is clearly detected only after cleavage 12 at a stage called the midblastula transition (23). In the presence of dactinomycin, embryos develop up to the midblastula transition, whereas this development is blocked by puromycin or cycloheximide (3, 20). Similar results have been obtained for maturation, which is independent of new transcription but requires de novo translation (15). Qualitative analysis of the proteins synthesized in oocytes (stage VI), in unfertilized eggs, and in embryos has shown that during maturation and after fertilization, new proteins appear but others are no longer synthesized (5, 19, 29). This suggests that the synthesis of specific gene products necessary for maturation, for the metaphase block in unfertilized eggs, and then for rapid proliferation is regulated at the translational level from the bulk of maternal mRNA. By differential screening of a cDNA library prepared with poly(A)+ RNA from unfertilized eggs, we isolated 11 nonoverlapping sequences which undergo either adenylation or deadenylation after fertilization (26). Nine of them which constitute the Eg family correspond to RNAs which are specifically deadenylated (24-26) and released from polysomes after fertilization (24, 26). In a previous work, we have shown that Egl has very high homology to p34cdc2 but is clearly functionally different from the kinase subunit of the maturation-promoting factor (24). In the present report, we describe the characteristics of EgS, which clearly belongs to the kinesine-related protein family. Kinesin was first discovered during studies of organelle transport in giant squid axons (1, 30). Subsequently, kinesin has been found in a variety of organisms and cell types, including avian and mammalian neuronal tissues (4, 30), sea urchin eggs (28), cultured cells (22), and Drosophila melanogaster (27). Biochemical studies of kinesin protein revealed that it is a tetramer consisting of two heavy and two light chains (2, 14). The gene that codes for the drosophila heavy chain has been cloned and characterized (33, 34). *
More recently, genes that encode kinesin-related proteins were identified by genetic criteria in D. melanogaster (7, 35) or by molecular genetic critera in Saccharomyces cerevisiae (18), Aspergillus nidulans (8), D. melanogaster (16), and Schizosaccharomyces pombe (11). The KAR3 mutant of S. cerevisiae is defective in karyogamy (18). Mutations in the bimc gene of Aspergillus nidulans prevent spindle pole body separation and nuclear division (8). In S. pombe, cut7+ is referred as a gene involved in spindle formation (11). The product of the drosophila claret gene is clearly implicated in chromosome segregation and is active in meiosis (7). The nod gene is required for distributive segregation of nonexchange chromosomes during meiosis in D. melanogaster (35). The sequencing strategy, the nucleotide sequence, and the predicted amino acid sequence of Eg5 are shown in Fig. 1A and B. The cloned EgS cDNA is 3,651 nucleotides long and contains 193 nucleotides of 5'-flanking sequence, an open reading frame of 3,180 nucleotides, and a 3'-untranslated region of 278 nucleotides which contains a potential polyadenylation signal (AAUAAA) at nucleotide position 3614 to 3619. The open reading frame codes for a predicted polypeptide 1,060 amino acids long with a relative molecular mass of 119,332 Da. The secondary structure predicted (10, 32) for EgS protein is shown in Fig. 1C. The predicted EgS protein has two globular domains separated by an a-helical region, which is a characteristic of all known kinesin and kinesinrelated proteins. Like all of these proteins, EgS also possesses a putative motor domain, which is shown in Fig. 2 (from amino acids 1 to 358). Figure 2 also shows the similarities of the motor domain encoded by EgS (defined as amino acids 1 to 358, corresponding to the end of Kar3) to the proteins encoded by bimc (8), the drosophila kinesin heavy chain gene (34), cut7+ (11), claret (7), and KAR3 (18), which are, respectively, 53.3, 49.7, 38.9, 29.9, and 28.6%. At amino acid positions 92 to 107, Eg5 possesses a putative ATP-binding site (IFAYGQTXXGKTXTM) which is conserved in all five sequences. As in the drosophila and squid kinesin heavy chains (13, 34) and the nod (35)-, bimc (8)-, and cut7+ (11)-encoded proteins, the proposed motor domain of Eg5 is located at the amino terminus. In contrast, in the KAR3 (17)- and claret (7, 33)-encoded proteins the mechanochemical domain is situated in the carboxy-terminal region. The sizes of the proteins encoded by EgS, bimc, cut7+, and the drosophila heavychain gene are in the same range, at 1,060, 1,211, 1,073, and
Corresponding author.
t Present address: Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545. 3395
3396
NOTES
A
MOL. CELL. BIOL.
E
_
i_ 0
*6
o
0
*
>
~ 500 bp
B
gottggctgtggctgtcggaagcgcgattttcaaatgtgtctgctgcgaggag3agccgt 60
gcttgcggaacgattttaacgcttggctgtagg4agtgggatcttcgcgctctggttact
120
tcaactgtctctgctcggggaagggaatcgtgactgcttattggagataactatgtcttc 180 ccaaaattcttttATGGCCAGCAAGAAGGAGGACAAAGGGAAAAACATCCAGGTTGTGGT 240 M
A
S
K
K
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D
K
G
K
N
I
0
V
V
v16
CCGATGCCGGCCCTTCAATCAGTTGGAACGCAAGGCGAGCTCTCACTCTGTGTTGGAATG 300 N
C
R
P
F
N
Q
L
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A
S
S
S
H
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C 36
TGATTCTCAGAGGAAAGAAGTCTATGTTCGGACCGGAGAAGTAAACGACAAACTGGGAAA 360 0
S
D
R
K
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Y
V
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N
K
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GAAAACCTATACGTTTGACATGGTGTTTGGGCCTGCAGCCAAGCAAATTGAGGTGTACAG T
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F
0
N
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420 R 7N
0
1 136
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AAGTGTTGTGTGCCCCATCTTGGATGAAGTTATTATOGGATATAACTGCACAATCTTTGC 480 S V V C P I L D E V I N G Y N C T I F A 96 GTATGGGCAGACTGGCACAGGCAAGACCTTTACTATGGAAGGGGAAAGATCATCAGATGA 540 Y K 0 T G T G K T F T M E G E R S S 0 EK 16 AGAGTTTACCTGGGAACAGGACCCTCTGGCAGGAATTATTCCTCGAACGCTGCATCAGAT 600 F
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N
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L
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W
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ATTTGAGAAGCTCTCTGAGAACGGCACAGAGTTTTCGGTGAAAGTCTCTCTCCTGGAAAT 660 F
E
6
N
E
CTACAACGAAGAACTCTTTGATCTGTTAAGCCCATCTCCCGATGTTTGGGAAAGGCTGCA Y
6
AATGTTCGATGATCCTCGGAACAAAAGAGGAGTCATTATCAAAGGGCTGGAGGAGATATC 780 F
H
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D
P
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N
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TGTGCATAACAAGGATAGGWTGTACCATATATTGGAGAGAGGTGCAGCCAGGAGGAAAAC 840
V 6 N K D E V Y N I L E R G A A R R K T 216 TGCTTCTACTCTGATGAATGCATATTCCAGTCGATCCCATTCCGTGTTCTCGGTCACCAT 900 A S T L 6 N A Y S S R S N S V F S V T I 236
TCACATGAAAGAAACTACTGTTGATGGAGAAGAGCTTGTGAAGATCGGGAAGCTGAACTT 960 H
K
M
E T
E
V
L
V
K
I
G
K
L
N
L 256
GGTGGATCTGGCGGGAAGTGAAAATATTGGTCGTTCTGGAGCTGTGGACAAAAGAGCACG V D L A G S E N I G R S G A V 0 K R A R CGAGGCTGGAAATATCAACCAATCCCTGCTCACCTTGGGCAGGGTAATAACTGCTCTGGT E A G N I N 0 L L T L G R V I T7 A V GGAGAGAACTCCTCATATTCCATACAGAGAATCCAAACTCACAAGGATCCTGCAGGATTC E R T P H I P Y R E S K L T R I L 0 D S TCTTGGAGGCAGGACAAAAACCTCTATCATTGCAACAGTGTCACCTGCATCCATTAATCT L
G
G
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N
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L
GGAGGAAACTGTGAGCACTTTGOACTATGCCAACAGAGCAAAGAGTATTATGAATAAGCC E
T
E
V
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0
Y
A
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N
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AGAGGTTAACCAGAAACTTACAAAGAAGGCACTCATCAAGGAGTACACCGAAGAGATTGA E
V
N
0
K
L
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6
K
A
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L
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E
T
Y
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1020 276
1080 296 1140 316 1200 336 1260 356 1320
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V
GCGACTTAAGAGGGAACTTGCTGCAGCCAGAGAGAAGAATGGAGTTTATTTATCCAGTGA 1380 R L K R E L A A A R E K N G V Y L S S E 395 GAACTATGAACAATTACAGGGAAAAGTTCTGTCTCAAGAGGAGATGATTACGGAGTACAC 1440 N Y E 0 L 0 G K V L S 0 E E M I T E Y T 416 TGAGAAAATAACTGCCATGGAGGAGGAACTGAAAAGCATCAGTGAGCTGTTTGCTGACAA 1500 E K I T A M E E E L K S I S E L F A D N436 TAAGAAGGAGCTGGAGGAGTGCACCACCATCCTTCAGTGCAAGGAGAAGGAACTGGAGGA 1560 K K E L E E C T T I L 0 C K E K E L E E 456 AACACAGAACCACCTGCAAGAATCAAAGGAACAGTTGGCTCAGGAATCATTTGTGTGTC 1020 T 0 N N L 0 E S K E 0 L A 0 E S F V V S 476 TGCCTTTGAGACTACAGAAAAGAAGCTTCACGGCACAGCAAACAAGTTATTGAGCACAGT 1680 A F E T T E K K L N G T A N K L L S T V A96 GAGGGAGACAACAAGAGATGTGTCGGGTCTCCATGAGAAATTGGACAGAAAAAAAGCTGT 1740 R
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T
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AGACCAGCACAACTTTCAGGSTCATGAGAACTTTGCGGAACAAATGGATAGACGCTTCAG
SIG
1800 S 536
TGTGATTCAGCGAACTGTGGATGATTACAGTGTGAAGCAGCAAGGCATGCTGGATTTCTA 1660 I
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TACAAACTCCATCGACGACCTTCTGGGCGCCAGTTCCTCTCGACTGAGTGCAACTGCCTC T
N
S
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D
L
L
G
A
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S
S
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1920 S 576
TGCTGTTGCCAAGTCCTTTGCCTCGGTGCAGGAAACTGTAACAAAGCAAGTGTCGCACAG 1980 V
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0
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TGTTGAGGAGATACTGAAGCAGGAAACCTTGTCTTCCCAAGCTAAAGGTGATCTGCAGCA 2040 V E I L K 0 E T L S S 0 A K G D L 0 0616
GCTTATGGCTGCGCATCGCACAGGACTGGAAGAGGCACTAAGGTCCGATTTGCTTCCAGT 2100 L
A
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R
T
G
L
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L
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D
S
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TGTGACTGCTGTTTTGGACCTAAATTCTCATTTGAGTCATTGCTTGCAGAACTTCCTAAT 2160 V
N
0 N F L I 656 TGTGGCCGACAAGATTGATTCTCATAAAGAAGATATGAATTCCTTCTTCACCGAACATTC 2220 V A 0 K I 0 5 H K E 0 M N S F F T E H S 676 TAGATCGTTGCACAAACTTCGGCTGGATTCTAGTTCTGCCTTGTCCAGTATTCAGTCAGA 2280 R S L 0 K L R L 0 S S S A L S S I 0 S E 696 ATATGAAAGCCTAAAAGAGGACATTGCSACAGCTCAGTCCATGCATTCAGAGGGGGTGAA 2340 Y E S L K E 0 I A T A 0 S M H S E G V N 716 TAATCTTATCAGTTCGCTACAAAACCAGCTGAACTTGCTTGGCATGGAGACCCAACAACA 2400 N L I S S L 0 N 0 L N L L G M E T 0 0 0 736 ATTTTCTGGATTTCTGTCCAAAGGAGGAAAGCTGCAGAAGTCTGTGGGGAGTCTGCAGCA 2460 F S G F L S K G G K L 0 K S V G S L 0 0 756 AGATCTGGACTTGGTTTCCTCGGAAGCCATAGAATGTATCTCTTCCCATCACAAOGAAGCT 2520 D L D L V S S E A I E C I S S 0 14 K K L 776 T
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L
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N
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H
C
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CGCTGAGCAGTCTCAGGACGTGGCTGTGGAGATCAGGCAGCTGGCAGGCTCTAATATGAG 2580 0
E
A
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0
0
V
L
V
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I
R
0
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A
G
S
N
M5 796 2640 I S 61
CACCTTGGAGGAGTCCAGCAAACAGTGTGAGAAGCTCACCAGCAGTATCAATACCATCTC T
E
L
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5
S
K
0
C
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K
L
T
S
I
S
N
T
TCAGGAAAGCCAGCAGTGGTGTGAAAGTGCCGGCCAGAAGATTGACTCTGTTTTAGAGGA 2700 Q E S 0 0 W C E S A G 0 K I 0 S V L K E 636
GCAAGTGTGTTACCTGCATTCCAGCAAAAAGCATCTCCAGAACTTACACAAGGGTGTAGA 2760
0 V C Y L H S S K K H L 0 N L H K G V E 656 AGATAGCTGTGGATCATCGGTOGTAGAAATTACCGACCGTGTGAACGCACAGCGCCAGGC 2620 D S C G S S V V E I T D R V N A 0 R 0 A 876 TGAGGAGAAGGCATTAACCAGTTTGGTGGAA TCAGGGATGACCAGGAGATGGTTGG 2880 E
E
K
A
L
T
S
L
V
E
0
V
0
V
R
0
0
D
E
M
v
H
S
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196
AGAACAGCGCtTGGAACTCCAAGAACAAGTACAGAGCGGCCTGAACAAAGTACATAGCTA 2940 0
E
R
L
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L
0
E
0
S
G
L
K
N
V
Y 916
CCTCAAGGAGAACTCAGGAATGATGTCCCAACAGGTACAACGCCCCAGAGGAGGGATTA 3000 L K E E L R N 0 V P T G T T P 0 R R 0 Y936
CGCGTACCCATCTTTGCTTGTTAAAACAAAACCCAGGGATGTGCTGTTGGAGCAGTTTAG A Y P S L L V K T K P R D V L L E 0 F R GCAACAGCAGCAAGAATATCTGGAATCCATATCCAGCGTGATCTCTGAGGCTGTGGAACC 0 0 0 0 E Y L E S I S S V I S E A V E P GCCAGTGGAGCAGGATTCTCTAGAAGATGAGCCACCGGTTGCAGTTAATGACAGCGTCAT P V E 0 D S L E D E P P V A V N D S V 1 CAGTGAGAGATCCTGCATTGATCTCAGTATGACTTGTCAGGAGAAAGGAGGAATTCGATT S E R S C I D L S H T C 0 E K G G I R F CTTCCAGCAAAAGAAAGCACTTCGGAAGGAAAAGGAAAACCGGGGAAATACAACACTTTT F 0 0 K K A L R K E K E N R G N T T L L GGAGAGATCTAAAATCATGGACGAGGTGGACCAAGCTCTTACGAAATCTAAACTTCCCCT E R 5 K I H 0 E V 0 0 A L T K S K L P L GCGAATGCAGAACtga agcacttgcttaacccScatgatgatgtgcggctttgtctOtct 6 0
R
N
3060
956 3120 976
3180
996 3240 1016 3300 1036 3360
05S
3420 1060
gtgcagaaa,lttga.tacatttttaactgtttstgStatccgggtgttgcgtatgSagaa
3460
aattttattcatcaataaatattcccttttctataaaaaoaaaaaaaaaa
3651
ettttacttatcttggttatatatatgtgtgtctatcgtcacttgtatgtcaaatattaa 3540 aaatactttctaaagtagaacttctcaggctttacgagttagttatggaaaagtStattt 3600
c
II§ 1 1 I J LW IJ IfI
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GOR
HELICES
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I
0
500
975 amino acids, respectively. There is no obvious similarity between the amino acid sequences of Eg5 and the drosophila heavy chain in the carboxy-terminal regions of the molecules. By contrast, as shown in Fig. 2B, significant similarity was found among the proteins encoded by Eg5, cut7+, and bimc. In particular, from amino acids 911 to 956, more than 35% of the Eg5- and bimc-encoded amino acids are identical and more than 50% are of the same family. The similarity extends beyond sequence comparisons; in particular, the structures of the central regions of the proteins encoded by Eg5, bimc and cut7+ are very closely related, which could suggest similar functions for these gene products. In a previous work (26), we have shown that Eg5 RNA translation is regulated during very early development. In eggs, the RNA is found in both the poly(A)+ fraction and polysomes. After fertilization, Eg5 RNA is deadenylated and concomitantly released from the polysomes. To know when Eg5 RNA appears and when it is adenylated during oogenesis, we performed Northern (RNA) analysis of total RNAs extracted from oocytes (stages I to VI), eggs, and premidblastula transition embryos by using a 32P-labeled Eg5 cDNA probe. Considering that a fixed quantity of total RNA (10 Rg) was used and that rRNA constitutes a progressively larger proportion of the total RNA during oogenesis (i.e., 25% in stage I oocytes versus 90% in stage VI oocytes), the decrease in the autoradiogram signals observed in Fig. 3A reflects the dilution of these transcripts in the pool of total RNA. Taking this into account, quantitation of autoradiograms from these Northern analyses confirmed that the number of these transcripts per oocyte or embryo was approximately constant from stage III of oogenesis to the midblastula transition (Fig. 3B). Closer examination of the data (Fig. 3A) shows that Eg5 transcripts appear to be larger in eggs than in either oocytes or embryos, suggesting that the RNA is adenylated only in eggs. When poly(A)+ and poly(A)- RNAs were separated from the total RNAs extracted from oocytes at different stages and analyzed by Northern blotting with Eg5 as the probe, we never found Eg5 in the poly(A)+ fraction (Fig. 3C). Moreover, in its 3'untranslated region (at nucleotide positions 3430 to 3436), Eg5 RNA possesses a sequence motif [UUUU(A)AU] similar to those shown to be necessary for maturation-specific adenylation of Xenopus mRNA (9, 17). This supports the hypothesis that during maturation Eg5 translation is regulated through adenylation, as recently reviewed (12, 31), and also suggests that the protein is present in eggs but not in oocytes. Antibodies against bovine kinesin reveal a pair of 120-kDa polypeptides in a crude Xenopus egg extract (28). After fertilization, Eg5 RNA is no longer detected in polysomes, suggesting that its further translation is not necessary for early development. This is in agreement with the notion that the 12 rapid cell divisions which follow fertilization
1000
FIG. 1. Nucleotide sequence of Egl cDNA and predicted amino acid sequence of the protein. (A) Restriction map and sequencing strategy. The arrows denote the extent and direction of sequence reading of each fragment. (B) Nucleotide and amino acid sequences. The nuclear polyadenylation signal is indicated by solid underlining (nucleotide positions 3614 to 3619). The putative cytoplasmic polyadenylation sequence specific for maturation is indicated by broken underlining (nucleotide positions 3430 to 3436). (C) Secondary structure prediction for Eg5 protein. The secondary structure of Eg5 protein predicted by the method of Garnier et al. (10) is shown. Regions predicted to be a turns (GOR turns), a helices (GOR alpha helices), or ,B sheets (GOR beta sheets) are indicated by the elevated segments.
VOL. 11, 1991 Eg5
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FIG. 3. (A) Northern blot analysis of total RNAs extracted from eggs (UFE), and embryos 4, 6, 8, and 10 h after fertilization. Samples of RNA (10 ,ug) were separated on agarose gel containing 6% formaldehyde and blotted onto nylon membrane (Hybond [Amersham]). Purified inserts were 32P labeled with random primers to a specific activity of 5 x 108 cpm ,Lg-1. Hybridization was carried out in 50% formamide-1% sodium dodecyl sulfate-10 x Denhardt's solution-10% dextran sulfate-1% PPi-1 M NaCl-0.05 M Tris-HCl (pH 8) at 42°C overnight. Filters were extensively washed in 2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.5% sodium dodecyl sulfate at 65°C. (B) Quantitative analysis of Eg5 RNA during oogenesis and very early embryogenesis. MBT, midblastula transition. (C) Total RNAs were extracted from oocytes (stages I and II, III and IV, and V and VI) and unfertilized eggs (UFE). Poly(A)+ was separated from poly(A)by oligo(dT) chromatography. Samples [10 pLg of total RNA, 10 ,ug of poly(A)- RNA, and 200 ng of poly(A)+ RNA] were analyzed by Northern blotting as described for panel A.
oocytes (stages I to VI), unfertilized
DHCLALAESQKQGVNLEVQ*JRLLQKVKEH 863
EITDRVNAQRQAEEEALTSLVEC6DDQEMVGORL4s DSYSSIEGRVEHLTGRNNQFQQEATNHHATLEIIA; 909 903
NNDNLIDSIETPHTELQK.ITDIeLKGTTSLANHTNFU 922 NRF1;*?FCPR*MLEQ. QQQ1 961
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3397
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FIG. 2. The predicted amino acid sequence of the motor domain of Eg5 is aligned with those encoded by Aspergillus bimc, S. pombe cut7+, the drosophila kinesin heavy-chain gene, saccharomyces KAR3, and drosophila claret. The numbers of the residues at which the bimc, cut7+, claret, and KAR3-encoded proteins extend in the amino-terminal direction and the drosophila heavy chain extends in the carboxy-terminal direction are in parentheses. Alignment of the carboxy-terminal parts of the Eg5-, bimc- and cut7+-encoded molecules is also shown. Identical residues are boxed. The central regions of the Eg5-encoded protein (from amino acids 366 to 535) and the bimc-encoded protein (and also the cut7+-encoded protein, to a lesser extent) show conserved amino acids (dots) spaced by six amino acids. Most of these conserved amino acids are leucines.
could only be under the translation control of cyclins, as suggested by in vitro experiments using egg extracts (21). Moreover, the fact that Eg5 RNA was not detected in adult tissues (26) suggests that it is involved in cell proliferation. Taking these points into account, and also the similarity among EgS, cut7+, and bimc, one can postulate that Eg5encoded protein is involved in nuclear division and may be the counterpart of bimc-encoded protein in xenopus. Nucleotide sequence accession number. The sequence described here has been assigned EMBL accession no. X 54002. We are grateful to Michel Kress and H. B. Osborne for very helpful discussions, to Chris Ford and Sharyn Endow for critical reading of the manuscript, and to F. de Sallier Dupin for preparing the manuscript. This work was supported by the Association pour la Recherche sur le Cancer, the Ministere de la Recherche et de la Technologie, la Ligue Departementale d'Ille et Vilaine de Recherche contre le Cancer, and la Fondation pour la Recherche Medicale.
3398
NOTES
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19. Meuler, D. C., and G. M. Malacinski. 1984. Molecular aspects of early development. Plenum Publishing Corp., New York. 20. Miake-Lye, R., J. W. Newport, and M. Kirschner. 1983. Maturation promoting factor induces nuclear envelope breakdown in cycloheximide arrested embryos of Xenopus laevis. J. Cell Biol. 97:81-91. 21. Murray, R. W., and M. W. Kirschner. 1990. Dominoes and clocks: the union of two views of the cell cycle. Science 246:614-621. 22. Neighbors, B. W., R. C. Williams, Jr., and J. R. McIntosh. 1988. Localization of kinesin in cultured cells. J. Cell Biol. 106:11931204. 23. Newport, J., and M. Kirschner. 1982. A major developmental transition in early Xenopus embryos. I. Characterization and timing of cellular changes at the midblastula stage. Cell 30:675686. 24. Paris, J., R. Le Guellec, A. Couturier, K. Le Guellec, F. Omilli, J. Camonis, S. MacNeill, and M. Philippe. 1990. Cloning by differential screening of a Xenopus cDNA coding for a protein highly homologous to cdc2. Proc. Nati. Acad. Sci. USA 88: 1039-1043. 25. Paris, J., H. B. Osborne, A. Couturier, R. Le GuelHec, and M. Philippe. 1988. Changes in the polyadenylation of specific stable RNA during the early development of Xenopus laevis. Gene 72:169-176. 26. Paris, J., and M. Philippe. 1990. Poly(A) metabolism and polysomal recruitment of maternal mRNAs during early Xenopus development. Dev. Biol. 140:221-224. 27. Saxton, N. M., M. E. Porter, S. A. Cohn, J. M. Scholey, E. C. Raff, and J. R. McIntosh. 1988. Drosophila kinesin: characterization of microtubule motility and ATPase. Proc. Natl. Acad. Sci. USA 85:1109-1113. 28. Scholey, J. M., M. E. Porter, P. M. Grissom, and J. R. McIntosh. 1985. Identification of kinesin in sea urchin eggs and evidence for its localization in the mitotic spindle. Nature (London) 318:483-486. 29. Smith, R. C. 1986. Protein synthesis and messenger RNA levels along the animal-vegetal axis during early Xenopus development. J. Embryol. Exp. Morphol. 95:15-35. 30. Vale, R. D., T. S. Reese, and M. P. Sheetz. 1985. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42:39-50. 31. Wickens, M. 1990. In the beginning is the end: regulation of poly(A) addition and removal during early development. Trends Biol. Sci. 15:320-324. 32. Wolf, H., S. Modrow, M. Motz, B. A. Jameson, G. Hermann, and B. Foertsch. 1988. An integrated family of amino acid sequence analysis programs. Comput. Appl. Biosci. 4:187-192. 33. Yang, J. T., R. A. Laymon, and L. S. B. Goldstein. 1989. A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses. Cell 56:879-889. 34. Yang, J. T., W. M. Saxton, and L. S. B. Goldstein. 1988. Isolation and characterization of the gene encoding the heavy chain of Drosophila kinesin. Proc. Natl. Acad. Sci. USA 85:1864-1868. 35. Zhang, P., B. A. Knowles, L. S. B. Goldstein, and R. S. Hawley. 1990. A kinesin-like protein required for distributive chromosome segregation in Drosophila. Cell 62:1053-1062.
