Sequence of a cDNA encoding a mouse cyclin B protein

Gene, 108 (1991) 315-316 0

1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

315

0378-l 119/91/$03.50

06192

Sequence of a cDNA encoding a mouse cyclin B protein * (Cell cycle; mitosis;

MUS musculus; recombinant

DNA;

homologies

to human

and Xenopus genes)

Gary D. Paternoa and Karen M. Downs b u Terry Fox Cancer Research Laboratories, Faculty of Medicine, Memorial University,St. John 2, Newfoundland (Canada) and b Developmental Biology Unit, Imperial Cancer Research Fund, Department of Zoology. South Pa& Road, Oxford University, Oxford, (England) Tel. (44)-86559977 Received by D.T. Denhardt: 27 August 1991 Accepted: 20 September 1991 Received at publishers: 23 September 1991

SUMMARY

We report here the nucleotide sequence of a cDNA encoding a mouse (MUS musculus) cyclin B protein. protein shows 84%, 66 y0 and 49% similarity with human cyclin, Xenopus cyclin B 1 and B2, respectively.

The cyclins are a family of phosphoproteins which have been conserved through evolution from yeast to man and which are crucial components of a protein kinase cascade regulating a cell’s entry into and exit from the M phase of the mitotic and meiotic cell cycle (Hunt, 1989; Lewin, 1990). As part of a study to identify markers of germ-cell lineages and their development during mouse embryogenesis we have isolated a cDNA encoding a murine homologue of the cyclin B proteins. Oligo primers were synthesized based upon conserved regions within the human and Xenopus cyclin B sequences. These primers were used to amplify, by PCR, a predicted 230-bp fragment from first strand cDNA synthesized by reverse transcription of poly(A)’ RNA isolated from PI9 murine embryonal carcinoma (EC) cells (McBurney and

Correspondenceto: Dr. G.D. Paterno, University,

St. John’s, Newfoundland

Tel. 709-737-7012; * On request conclusions Abbreviations:

of Medicine, AlB 3V6.

Memorial

Fax 709-737-7010.

the authors reached

Faculty (Canada)

will supply detailed

experimental

evidence

for

in this Brief Note.

aa, amino

oligo, oligodeoxyribonucleotide;

acid(s);

bp, base

pair(s);

PCR, polymerase

nt, nucleotide(s);

chain reaction.

The deduced

Rodger, 1982). Subcloning and sequencing of this fragment revealed significant similarity to human and Xenopus cyclin B sequences. This 230-bp fragment was used as a probe to isolate full-length cyclin clones from a P19 EC cell cDNA library. Several clones were isolated and the largest ofthese, called pC5237, was completely sequenced and found to encode a murine homologue of the human and Xenopus cyclin B (Fig. 1). The cDNA is 1481 nt in length and includes a 88-bp 5’-untranslated region preceeding an open reading frame of 1290 nt beginning with an ATG within an initiation site predicted by Kozak (1986) rules. The 3’-untranslated region is 99 nt long, ending with a poly(A) tail contains a polyadenylation consensus signal (5’-AATAAA) located 24 nt from the 3’ end. The 430 aa polypeptide, deduced from translation of the cDNA, shows an 84% identity with the human cyclin B protein (Fig. 2) and 66% and 49% identity with the Xenopus cyclin Bl and B2 sequences, respectively (data not shown). The deduced aa sequence displays several motifs common to the cyclins including the highly conserved ‘cyclin box’ (aa 189-34; Hunt, 1989), a short region of homology to the adenovirus ElA transcription factor (aa 87-105; Pines and Hunter, 1989) and the cyclin destruction box (aa 42-50; Glotzer et al., 1991).

n

R AK K ACT T CCT P CTA L CM E CAA E CCG c c A GCA GTG AGT A” s TAT CTC AM V” K AW CCA AM

4.32 115 486 133 540 151 594 161 648

D I A L R P L E E E 0 S” R P K TAC CT* CM CCT CCT CAA CTG ACT GCA MC ATG *CA CCT AK CTC ATT CAC TCC vl.PCRE”TCNnRAlLlDW CT* ATA CAG GTT CAG ATC ALA TTT ACC CTG CTT CAG CAC ACC AK TAC AK ACT

187 702 2°C 756

AK N CGT R” CCT * CTC L CCA P CCT P CCT P CCT PA WC 0” WT

KC MA T I: GTC CCT P” CTT GCA L c AM ACG I: R AM CCT I( P” WG CCT E P ATT TTC I L clx GM E GTA WC D *,c TAT

v

ATT MC I N CTC AU TV GAC ATT D I CM cc* E A CT* WG E CAA CCT E P GTT CAT ” *D CAG TAT E Y GCA CAT * 0 GCT TAT

CCA CA* A E CTT ACT T GGT MT c N AM KG K T AK GTG I(” CAA CTT E L AAT ccc N P KG TGT P c ACT CCC

s

c cc*

CTC

MC ccc K A GCT TCC * s GTC ACC

s CCT ACT c T CTC ET

c C*T H”

CT,

cc*

AGC

s P s CAC CCT TTC PA f CCT CAC ox A 0 P CAA CTC CAC

n I( F R L L a E T n v n T CGG TTC ATC CAG AK ACT TGT GTC ccc MC MC ATC AT* R F n P N s C” P I( I( II I.?,, ACC ccc ATC TTT *TT cc* AK AM TAT GM CAC AK TAC T A n F I * s K Y E cl n v GAC TTC ccc TTT GTC ACT MC MC KC TAC ACT AAG CAC CAC I\TC AAG ATT CTC ACA CT, CTC MC TTC KC CTG GGT E n I( I L R ” L N F s L G CAC TTC CTC CGT ACA cc* TCT AM CTC CGA WIG CTT CAC CTG ccc AAA TK CTC ATG CAL? CTC KC ATC CTC CAC TAC L A K v L n E L s n L D v CCT CCT TCC AGC GCA TTT TCT ccc CCT TTC TCC TTA ccc A P s R A F s G A F c L A AK CGT GAA TCC ACA CCA ACT CTG UC UC TK CTA KC CTG CTT CCT CTT ATG LAG UC

CTG GCT MG

AAT

GTA CTC

CTC ACA AAC CIC ATC *CT WC MC MC MG TAT GCA GCA I_ T K H n TV 1: N K v A * AK *cc KC UC GCA CAG CTC UC KT ACA CAT GTT CAG I s T L A P L N c T H” a UC AtA AAG GCA TAIA CTC CM TAG ACT *CA TCT GCA WIG CCT CT* CAA Glx CTC ACG A

Fig. 1. The nt sequence aa sequence.

TIC

of the cloned

CTA

ccc

TCT TTA CTT CCT CTT CM

mouse

cyclin B cDNA

The aa are below the nt sequence

with the second nt ofeach are indicated

signal is underlined.

in the right margin.

7 162

~LRVT~TKINAENKAKVSMACAKRVWNTMSKPGL~RTALGDIGN . . . . . . . .. . . . . . . . .. .. .. .. .. .. .. .. .. . ,....... . .:.:::::::::::::::

50

t!ALRVT~KINAENKAKIWACAKRVPTAPAATSKPCLRPRTALGDlCfl

50

25

KVSEELWTVPLKREAKTLGTCKClVKALPKPVEKVP------VCEPEVE :::: ::: .:.:.::: ::: : ::::.::::

216 42

270 6, 324

94 :

::

KVSEC%WUlFtlKKEAKPSATGKVlDKKLPKPLEKVWLVPVPWEP---

97

UEPEPEPELEHVREEKLSPEPILVMPSPSFtlETCGCAPAEEYPCQ4FS

79

378

..““”

97

:

:.::::::::::::

144

.‘.“.:: . . . .

.“” . . . . . .

:::::

VPEPEPEPEPEPVKEEKLSPEPILVDTASPSFMETSCCAPAEEDLCBAFS

147

DVILAVSDVDADSCADFNLCSEWKDIYAYLRQLEEEQsVFWKYLQCREV

194

DVILAVNDVDAEDCADFNLCS~KDIYAYLRQLEEEBAVRPKYLLGREV

197

::::::

. . . . . .. .. .. .. .. .. . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . ::::

TUWUILIDWLIQVCflKFRLLQETMYWVSIIDRFHWSCVPKt3lIQLV

............ .......................... ..............................................

244

......

...

TWlPAILIWLVQVWlKFRLLQETMWlTllDRFtl@WCVPKKMLQLV

247

CVTAMFIASKYECilYPPEIGDFAFVTNNlYTKHQlRC?lMKILRVlNFSL ::::::.....: . . . . . . . .. . . . . .. . . .. .. . .. .. .. .. .. .. .. .. .. . .. .. ..

223 818

294 :::

:

CVTAllFIASKYEMYPPEICDFAFVTDNlYTKHQIRQlMKILR4LNFGL

297

864

GRPLPLHFLRRASKVCEVDVRQHTLAKYLMELSllLDYCWHFAP?%AFSG :::::......... . . . . . . . . . .. .. .. .. . . . . . . . . . . . . . . . . . ..

159

9,s 277 972

344 ::

.:

GRPLPLHFLRRASKICWDVECHTLAKYLIlELTMLDYBlVHFPPSQlAAC

347

2’5

AFCLALEILWGWTPTLQHYLSYSEDSLLPWQHLAKNW?lVNCCLTKH . .. .. .. . . .. .. .. .. .. .. .. . .. . .. . . . . . . . . . . . . . . . . . . . .

,826 2 I7 10380

394 :::::

AFCLALKILCNGEWTPTLQHYLSYTEESLLPWIQHLAKNWHWQCLTKH

2.3

397

,134 ‘-17

,188 367 124* 3R5 ,296 4m ,350 12,

1404 110 ,458 ,481

MTVKNKYAASKHAK . .. . . . . .. . . .

I STIAQLNCTHVCWLSKAVTKA .

. .

:::

::

430

:.:::.:

MTVKNKYATSKHAKISTLPQLNSALVQDlAKAVAKV

433

Fig. 2. Comparison

and alignment

(upper)

(lower) cyclin B proteins.

and human

mouse sequence

of the mouse

The aa numbers

for the

are the same as in Fig. 1 and for human cyclin B are from

Pines and Hunter similar

of the aa sequences

(1989). Colons indicate

identical

aa and dots indicate

aa.

and the

and are aligned

codon. The hyphen in the aa sequence

the stop codon and the polyadenylation aa numbers

” T GCC ccc G A CCC *GA P R *CA me TV AM ccc K A CTT GCT I. * TCT CCT s P GCA TGT

v

L I 0” P GTG TCC *TT *.TT CAT “S I I D UC CTC CTC CCT CTA * L” C” CCA CCA CM ATA GCT PPEICDF*F”TNNTvTKH CAC *TC AW UC ATC Q I R P n ccc CCT CTG CCT CTC RPLPLHFLRRASKVGEVD CTC ACC CAG UC ACT ” R P H T CAC *TC CTG CAT TTT I3 n ” H F CTG GM *n CTT CAC LEILDNGEWTPTLPHVLS TAC AGT WA WC KC vSEDSLLP”M*HLAKN”” ATC crc MC TCT ccc nv N c c TCT AAC CAT CCT MC s K H A K MT TTC TCT MG ccc NLSrc*“TKATTG CCA CCA TCT ccc TAA AGG TTG TV. CTT

deduced

AAT N CCT A MA K” CT/I L CCT P” GM E TCT

A I. R AAG CTC ACT ATG WA I:” s n A AAC ccc ccc CTC Ala Y P c L R CA* GAG CTA CAC CCA E E L P A GCA AM CGT ACT CTT c I: c TV GM CCA WG GTG CM E P E” E AW CM GAG MG CTT R E E K I. ccc ATC GM AU TCT P n E T c TCT CAT CTA AK CTT s 0” f L AK CTC TCT ACT CM N L c s E a* cxc UC TO. CTT

indicates

The nt and

GeneBank/EMBL

acces-

sion No. is X58708.

REFERENCES Glotzer,

M., Murray,

the ubiquitin Hunt,

T.: Maturation

phase. ACKNOWLEDGEMENTS

We thank Dr. John Bell, University of Ottawa for providing the P19 Agtll library, and Drs. Tim Hunt, Cambridge University, and Peter Holland, Oxford University, for help in oligo design and discussions. This work was supported by the Imperial Cancer Research Fund and a grant from the National Cancer Institute of Canada to G.D.P.

Kozak,

A.W. and Kirschner,

pathway.

promoting

that modulates

(1986) 283-292. Lewin, B.: Driving substrates. McBurney,

M.W.: Cyclin is degraded

by

349 (1991) 132-137. factor,

cyclin

and the control

of M-

Curr. Opin. in Cell Biol. 1 (1989) 274-286.

M.: Point mutations

codon

Nature

define a sequence translation

the ceil cycle:

flanking the AUG initiator

by eucaryotic M phase

ribosomes.

kinase,

Cell 44

its partners

and

Cell 61 (1990) 743-752.

M.W. and Rodger,

B.J.: Isolation

ma cells and their chromosome

ofmale

replication

embryonal

patterns.

carcino-

Develop.

Biol.

89 (1982) 503-508. Pines, J. and Hunter cyclin mRNA

T.: Isolation

and protein

action with p34cdc.

of a human cyclin cDNA:

regulation

evidence

for

in the cell cycle and for inter-

Cell 58 (1989) 833-846.