Identification and characterization of the SMT3 cDNA and gene ...

Vol. 46, No. 4, November 1998 BIOCHEMISTRYand MOLECULAR BIOLOGYINTERNATIONAL Pages 775-785

I D E N T I F I C A T I O N A N D C H A R A C T E R I Z A T I O N OF THE SMT3 c D N A A N D G E N E ENCODING UBiQUITIN-LIKE PROTEIN FROM DROSOPHILA MELANOGASTER

Hung-Wen

H u a n g 1 , S t e p h e n C.-M.

Tsoi 2 , Y. H e n r y Sun 3 , and

1,2,4

Steven

S.-L.

Li

1

Department of Biological Sciences, National Sun Yat-Sen 2

University, Kaohsiung, Taiwan 80424, ROC,

,

Dlvislon of

Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA, and

3Institute of

Molecular Biology, Academic Sinica, Taipei, Taiwan 11529, ROC. Received June 18, 1998 ReceivedaRerrevision, August3,1998

A SMT3

cDNA encoding

ubiquitin-like

protein

from

Drosophila melanogaster was i s o l a t e d a n d sequenced. Drosophila SMT3 g e n o m i c D N A was a m p l i f i e d b y p o l y m e r a s e reaction,

a n d its n u c l e o t i d e

identical

to that of the cDNA,

intron acids

in its p r o t e i n

to y e a s t

indicating

c o d i n g region.

of Drosophila SMT3

identity

s e q u e n c e was

SMT3,

exhibited human

the a b s e n c e

SMT3A,

73%,

of

of 90 a m i n o

70% and 52%

SMT3B and SMT3C p r o t e i n

The n u c l e o t i d e

s e q u e n c e has b e e n d e p o s i t e d

database

accession

under

found to be

The s e q u e n c e

55%,

chain

in the G e n B a n k

no. AF053083.

4To w h o m all c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d NIH, RTP, NC27709, USA. Tel. 1 - 9 1 9 - 5 4 1 - 4 2 5 3 ; F A X 1 - 9 1 9 - 5 4 1 - 7 5 9 3 ; e-mail: L i S N I E H S . N I H . G O V

at NIEHS,

1039-9712/98/160775-11505.00/0 775

Copyright 9 1998 by Academic Press Australia. All rights of reproduction in any farm reserved.

Vol. 46, No. 4, 1998

BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

sequences,

respectively.

2.5 kb a n d

1.5 kb w e r e

embryo,

larvae

evolutionary

Two Drosophila SMT3 shown

and adult

to be a b u n d a n t l y

tissues.

relationships

expressed

The structural

among

of

in

and

ii SMT3 p r o t e i n s

Xenopus, Drosophila, nematode,

mouse,

transcripts

from human,

Arabidopsis, rice,

Cicer, a n d y e a s t w e r e also analyzed. A family SMT3,

human

The y e a s t

of u b i q u i t i n - l i k e

SMT3A,

SMT3

of m u t a t i o n s

SHT3B

gene was

in MIF2

to the A + T - r i c h

Studies

using

loss

Mif2p

missegregation,

regions

designated

as HSMT3)

independently

GMPI was

The m o d i f i e d

nuclear

with

PICI was

b y several PICI

protein

the o t h e r w i s e

associate (7,8).

(8),

multiprotein

RanGAPI,

protein

complex

linked

was as

(ii).

to the Ran of SUMO-I

to the n u c l e a r

also a p p e a r e d during

pore

to mitosis

the PML c o m p o n e n t

that is d i s r u p t e d

776

of

(formerly

and a t t a c h m e n t

with

CENP-

project

sentrin

apparatus

two

(3,4).

and d e n o t e d

(i0),

form of RanGAPI

to interact

SMT3B

groups

RanGAPI

spindle

the

at least

SMT3C p r o t e i n

(9), UBLI

cytosolic

the m i t o t i c shown

shares

from genome

cloned h u m a n (6). Human

that

microtubule

centromere

found to be c o v a l e n t l y

GTPase-activating

complex.

CDNA

(i) .

in c h r o m o s o m e s

and a b e r r a n t

identified

protein

DNA

showed

of active k i n e t o c h o r e s

(5). We have

isolated

as a s u p p r e s s o r

an e s s e n t i a l

mutants

results

yeast

discovered.

of c e n t r o m e r e

with mammalian

component

21

SUMO-I/GMPI

encodes

region

delay,

S M T 3 A gene was

(7),

identified

(2). The Yeast Mif2 p r o t e i n

chromosome

targets

CDEII

mitotic

C, an i n t e g r a l

SU-MO-I

which

function

of s i m i l a r i t y

Human

originally

including

was r e c e n t l y

temperature-sensitive

of y e a s t

morphologies

and SMT3C,

gene,

binding

proteins,

in acute

of

Vol. 46, No. 4, 1998

BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL

promyelocytic human

leukemia

RAD51/RAD52

proteins

DNA double-strand interact

(9). UBLI was

b r e a k repair

with Fas/APO-I

the o v e r e x p r e s s i o n

(i0).

S e n t r i n was

or TNF r e c e p t o r

and T N F - i n d u c e d

H e r e we report

the cloning

cell death

from human,

relationships

and

against

mRNA

D N A from the s t r u c t u r a l

of ii SMT3 p r o t e i n

sequences

Xenopus, Drosophila, nematode,

mouse,

Arabidopsis, rice, MATERIALS

to

(ii) o

cDNA,

of genomic

and

domains,

protection

Drosophila melanogater. We have also a n a l y z e d and e v o l u t i o n a r y

shown

i death

of a SMT3

and c h a r a c t e r i z a t i o n

with

in DNA r e c o m b i n a t i o n

of sentrin p r o v i d e d

both anti-Fas/AP0-1

expression,

involved

found to a s s o c i a t e

Cicer, and yeast.

AND METHODS

Isolation and characterization of SMT3 cDNA and gene. Human

SMT3C p r o t e i n

sequence

Drosophila EST database, AA264131)

was

and an EST

found to c o n t a i n

polymerase-chain-reaction sequence reverse

were:

CA)

(PCR) primers

(Boehringer

sec at 94~

annealing

sequence.

containing

30 sec at 62~

a n d 25 cycles

a n d elongation,

5 cycles

The

777

at 68~

two

and

cDNAs La

the E x p a n d a mixture

of

PCR c o n d i t i o n s

were:

(denaturation,

30

and elongation,

(denaturation,

2 min.

no.

(Stratagene,

for PCR w i t h

Mannheim)

the

on the E S T

The m i x e d

cDNA library

denaturation,

annealing,

at 68~

accession

based

T a q and Pwo D N A polymerase.

of 2 min.

to b l a s t

5'-CATGTCTGACGAAAAGAAGGGAGG-3'

w e r e u s e d as templates

thermostable

min.

homologous

from Drosophila adult

High Fidelity

1 cycle

forward

(GenBank

5'-GTGGCGCTCATAAGATTACTTAF-3'.

prepared Jolla,

was u s e d as q u e r y

2

30 sec at 94~ The P C R - a m p l i f i e d

Vol. 46, No. 4, 1998


fragment was sequenced and labeled using PCR-Digoxigenin (DIG) labeling kit

(Boehringer Mannheim).

This labeled DNA

probe was then used to screen the cDNA library, positive cDNA clone was isolated. indicated in Fig.

and a

Two additional PCR primers

1 were used to amplify SMT3 DNA using

templates of genomic DNA

(BIOS laboratories,

New Haven,

CT,

USA) and cloned SMT3 cDNA. The sizes of PCR-amplified fragments from SMT3 cDNA and genomic DNA were compared on 1.0% agarose gel. The purified DNA fragments amplified from these two templates were labeled with the Dye Terminator kit (Perkin-Elmer,

Foster City, CA, USA),

and both strands of the

amplified DNAs were completely sequenced using automatic DNA sequencer

(Applied Biosystem,

USA, model 377).

Northern blot analysis. The poly(A§

from embryo,

larvae and adult were obtained from Clontech

(Palo Alto, CA).

Approximately 2 ~g of each poly(A+)-RNAs were ran on a 1.2% formaldehyde agarose gel for 3 hours at i00 volts and transferred upward to a Hybond-N § membrane Science,

Cleveland,

(Amersham Life

OH) by capillary action for an hour. The

membrane was fixed by UV crosslinking and then hybridized with DIG-labeled probe solution.

(25 ng/ml)

in DIG Easy hybridization

Prehybridization and hybridization conditions were

strictly followed according to the Genius System User's guide (version 3.0) except the temperature was 45~

The specific

SMT3 transcripts were detected by DIG luminescent detection kit.

Analyses of structural and evolutionary relationships among SMT3 proteins. The complete amino-acid sequence of

778

Vol. 46, No. 4, 1998

BIOCHEMISTRY and MOLECULAR BIOLOGYINTERNATIONAL

Drosophila SMT3 CACGCCCGGCATTCGACGCTCCGCAAAAGAAAAAAAAACTTTTTTGACCACTTA GCAGC T T C A A C A A G C A A C C A A A A A A T C A A C A T G T C T G A C G A A A A G A A G G G A G G T M

S

D

E

K

K

G

G

8

GAGACCGAGCACATCAACCTGAAGGTCCTCGGCCAGGACAACGCCGTCGTCCAG E

T

E

H

I

N

L

K

V

L

G

Q

D

N

A

V

V

Q

26

TTCAAGATCAAGAAGCACACACCCTTGAGGAAGCTGATGAACGCCTACTGCGAC F

K

I

K

K

H

T

P

L

R

K

L

M

N

A

Y

C

D

44

CGTGCCGGACTCTC CATGCAGGTGGTGCGCTTCCGTTTCGACGGACAGCCCATC R

A

G

L

S

M

Q

V

V

R

F

R

F

D

G

Q

P

I

62

AACGAGAACGACAC TCCGACCTCGCTGGAGATGGAGGAGGGCGACACCATCGAG N

E

N

D

T

P

T

S

L

E

M

E

E

G

D

T

I

E

80

GTTTACCAGCAGCAGAC TGGTGGCGCTCCATAAGATTACTTAGTTAAGTTAGTT V Y Q Q Q T G G A P * AC TC CTC T T A C A A C T A C A C A C T T A A A A C A A A A A A G A A A A A A A A T A C A A G A A A A A CCACAAAAGCAAAAACACAACAACAACAACATGAAGAATCCAACAAACCAGGC C CTAAGAATCGATTGAATATGCTTTTAGTACAACTGTAGATTCTAAATGCGTCTG TGTGCGTTAATAACAAAAACATTTGCAGACAAGAAAATGGT Fig.

i. T h e n u c l e o t i d e

a n d d e d u c e d amino a c i d s e q u e n c e s

90

of

Drosophila SMT3 cDNA. The stop c o d o n T A A is i n d i c a t e d b y an asterisk. PCR p r i m e r s u s e d are underlined.

Two

Drosophila melanogaster SMT3 p r o t e i n was d e d u c e d from the cDNA and gene

sequences

(Genbank a c c e s s i o n a n d the G e n b a n k proteins SMT3B,

no. AF053083).

accession

are as follows:

L76416;

AF033353), nematode,

determined

SMT3C,

African

numbers

frog,

package

mouse,

Rice,

Cicer arietinum (AJ001901)

the m u l t i p l e

of the o r g a n i s m s

of other r e p o r t e d

The a m i n o - a c i d

alignment

program

Z97073);

Thale

cress,

Oryza sativa (X99608); Saccharomyces

a n d yeast, sequences

were aligned

in G E N E W O R K

b a s e d on the m e t h o d of Feng and D o o l i t t l e

structural

and evolutionary

UPGMA method

SMT3

Mus musculus (SMT3C,

Caenorhabditis elegans (U94830);

cerevisiae (U27233). using

The names

Xenopus laevis (SMT3C,

Arabidopsis thaliana (X99609); chickpea,

investigation

Homo sapiens (SMT3A, X99584;

Human,

X99586);

in this

relationships

were

f r o m the same c o m p u t e r p r o g r a m 779

computer (12).

analyzed

(13).

The using

Vol. 46, No. 4, 1998


RESULTS AND DISCUSSION

Characterization of SMT3 cDNA and gene. A Drosophila SMT3 cDNA clone was determined contains

a protein-coding and 3'

protein-coding

(226 nucleotides)

region,

fragments,

were determined

These results

indicated

coding region

of Drosophila SMT3 gene.

nos.

the absence

sequence

by two small

introns

that two SMT3

1.5 kb were expressed although in larvae

in embryo,

the transcript sample

(Fig.

represent Further,

2),

and their

to be identical.

It is of interest SMT3 gene

of Northern

transcripts

blot

of 2.5 kb and

larvae and adult

3). These size differences

transcripts

tissues, present

may be due

sites used or alternative

It is also possible

that they

of two similar

genes.

SMT3

one might wonder why the small SMT3 protein

amino acids requires

of 90

such large transcripts.

Amino acid sequence comparison among SMT3 proteins. sequence

that

is

of 1.5 kb is most abundantly

of the same SMT3 gene. different

DNA were

(14).

to either different polyadenylation splicing

regions

of 56 and 50 bp at coden

Northern blot Analysis. The results indicated

(85

of intron in the protein-

of nematode

22-23 and 56, respectively

analysis

(Fig.

5'

including

from SMT3 cDNA and genomic

of 463 nucleotides

interrupted

SMT3 cDNA

untranslated

found to be the same on 1.0% agarose gel

the protein-coding

This

sequence of 270 nucleotides,

i) . The sizes of PCR-amplified

sequences

of 581 nucleotides

(GeneBank accession no. AF053083).

nucleotides) (Fig.

isolated and its sequence

The

of 90 amino acids deduced from the Drosophila SMT3

780

Vol. 46, No. 4, 1998

BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL

1

2

3 Kb

bp

I

0.5

463~

Fig.

1.0

2. Size c o m p a r i s o n of P C R : a m p l i f i e d

fragments u s i n g

Drosophila genomic DNA and SMT3 cDNA as templates. Lane i, genomic D N A as template; template;

lane 2, SMT3 c D N A as

lane 3, size m a r k e r of 1 kb DNA ladder

(BRL Life

Technologies). I

2

3

Kb

2.5

1.5

Fig.

3. N o r t h e r n blot analysis

of Drosophila SMT3

transcripts. Lane i, larvae;

lane 2, adult;

781

lane 3, embryo.

Vol. 46, No. 4, 1998

BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL

~A 0 ~ 0 0 0 0 ~ 0 0 0 0 O

o

o

o

o

~

o

o

o

~

R -,4

o

~

Q

H

H

H

>

~

~

>

)

H

H

H

H

H

H

o

~

NNN

~

M Z

H

~

~

H H H H H H H H H H H

I

N

I

M

M

M

~

O

"~ Q

m

r ~

-~

c,q

~ .,-i 4J o

H 9

H

N

N N

Z

N 0

N

N

N

N N N

N

~

N

N N

~ 9

u,q o

N

N N N m

(1) o

-,-I ~

~1

9 m

o +J

~-~ N

-~ o

@

~

~

o

o

~

~

H o

~

o o 9

4J

o

N 9

~ ~

o

oh o

o~ Oh o

q3 R

~

~3

m

I I

l l

l l

~ ~

l l

l l

l l

~ ~

RR

m

m

H

RRR

m

m

H

~

~

~

H

CO Oh o

9 a

N N ~ N ~ N N ~ N

tn

~

x O

Om

~

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o'

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o

(.9

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-,--I o o

9

O~ -~ om m

rn

R R R R R R

.~

o ~l co

9 ~

o

-,~

.r-,i

q5

Vol. 46, No. 4, 1998


c D N A and gene aligned with

sequences

determined

the SMT3 p r o t e i n

sequences

Xenopus, Drosophila, nematode, yeast

available

sequence

gene e x h i b i t e d SMT3,

human

SMT3A,

respectively. sequences. positions

55%,

Human

There

acids 73%,

from human,

(Fig.

SMT3B and SMT3C p r o t e i n

among

Cicer a n d

4). The d e d u c e d

70% and 52% i d e n t i t y

and m o u s e

residues

ii SMT3

cDNA and

to the y e a s t

sequences,

SMT3C p r o t e i n s

these

was

mouse,

from the Drosophila SMT3

are 21 identical

compared

investigation

Arabidopsis, rice,

in the databases

of 90 amino

in this

have

identical

out of Iii

sequences.

The

Human SMT3A Human SMT3B

Drosophila SMT3 Human SMT3C Mouse SMT3C - -

Xenopus SMT3C Nematode SMT3 Arabidopsis SMT3

Rice SMT3 Cicer SMT3

Yeast SMT3

Fig.

5. S t r u c t u r a l

and e v o l u t i o n a r y

relationships

among

ii

SMT3 proteins. The g r a p h i c a l relationships methods

among

display

of structural

the ii SMT3 was o b t a i n e d

(13).

783

and e v o l u t i o n a r y using UPGMA

Vol. 46, No. 4, 1998

significant proteins


amino acid identity and similarity of these SMT3

among different

importance.

The SMT3C proteins

were reported However,

species

to exhibit

the exact

indicate

their

from human,

diverse

function(s)

functions

functional

mouse

and Xenopus

(7-11,

15-16).

of human SMT3A and SMT3B,

Drosophila, nematode and plant SMT3 proteins remain to be elucidated

experimentally.

these ii SMT3 proteins

Drosophila, nematode, analyzed method

The structural

from human,

(13).

Results

are presented

with human,

SMT3 are clustered

Cicer and yeast were

tree constructed using UPGMA in Fig.

is clustered with human SMT3A and SMT3B, is clustered

among

Xenopus,

mouse,

Arabidopsis, rice,

and the evolutionary

relationships

5. Drosophila SMT3

while nematode

mouse and Xenopus SMT3C.

into a separate group,

SMT3

Three plant

and the yeast

SMT3

is the out group. ACKNOWLEDGMENTS We thanks Drs. reading

Frank Johnson and Po-Chuen Chan

the manuscript.

This investigation

part by grants NSC85-2732-B-II0-002 from National

Science

Council

for

was supported

in

and NSC86-2313-B-II0-002

of Taiwan,

ROC.

REFERENCES i.

Meluh,

P.B.,

and Koshland,

D.

(1995)

Mol. Biol. Cell

6, 794-807. 2.

Brown,

M. T.,

Goetsch,

L.,

and Hartwell,

L.H.

(1993)

J.

Cell. Biol. 9, 2544-2550. 3.

Saitoh,

H.,

Tomkiel,

Maurer,

M.,

Rothfield,

Cell 70,

J.,

Cooke,

N.F.,

115-125.

784

C.A.,

Ratrie,

and Earnshaw,

H.

W.C.

III, (1992)

Vol. 46, No. 4, 1998


4.

Brown, M. T.

5.

Lapenta,

(1995)

Gene 160,

V., Chiurazzi,

A., Hanooka,

111-116.

P., van der Spek, P., Pizzuti,

F. and Brahe, C.

Genomics 40,

(1997)

362-

366. 6.

Mannen,

H., Tseng, H.-M., Cho, C.-L.,

(1996) Biochem. 7.

Mahajan, Melchior,

8.

Matunis,

Boddy,

C., Quan, T., Gerace

L., and

Common.

Cell 88, 97-107.

Coutava,

E. and Blobel, G.

(1996) J. Cell

14~7-1470.

P.S.

(1996)

ii. Okura.

R.K. and Chen, D.J. T., Gong,

C.-F.,

P.E., Comeaux,

(1996)

L., Kamitani,

Genomics

T., Wada,

Chang, H.-M. and Yeh, E.T.H.

15,

Immunol.

E. and

Oncogene 13, 971-982.

Z., Pardington-Purtymun,

Moyzis,

Wei,

178-180.

Res.

M.N., Howe, K., Ettkin, L.D., Solomon,

Freemont, i0. Shen,

(1997)

M.J.,

135,

Biol.

9.

F.

222,

Biophys.

R., Delphin,

and Li, S.S.-L.

J.C.,

36, 271-279.

T., Okura,

I.,

(1996) J.

4277-4281.

12. Feng, D.F. and Doolittle,

R.F.

(1987) J. Mol.

Evol.

25,

351-360. 13. Swofford,

D.L. and Olsen, G.J.

Systemics

501,

(Hillis, D.M. and Moritz,

Sinauer,

14. Choudhury, Res.

P.S. and Solomon,

Nishimoto,

C.

Eds.), pp. 411-

(1997

Biochem.

Biophys.

234, 788-791.

15. Howe, K., Willisaon,

H.,

in M o l e c u l a r

Sunderland, FLA.

B.K. and Li, S. S.-L.

Commmen.

16. Saitoh,

(1990)

E.

Sparrow, T., Mohun,

J., Boddy, N., Sheer, D., Freemont, (1998) Genomics 47, 92-100. D.B.,

Shiomi, T., Pu, R.T.,

T.J. and Dasso

8, 121-124.

785

(1998)

Current Biol.