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Shark (Scyliorhinus torazame) Metallothionein: cDNA Cloning, Genomic Sequence, and Expression Analysis Young Sun Cho, 1 Buyl Nim Choi, 1 En-Mi Ha, 1 Ki Hong Kim, 2 Sung Koo Kim, 3 Dong Soo Kim, 1 Yoon Kwon Nam 1 1 Department of Aquaculture, Pukyong National University, Busan 608-737, South Korea 2 Aquatic Life Medicine, Pukyong National University, Busan 608-737, South Korea 3 Biotechnology & Bioengineering, Pukyong National University, Busan 608-737, South Korea Received: 22 April 2004 / Accepted: 7 October 2004 / Online publication: 4 June 2005 Abstract Novel metallothionein (MT) complementary DNA and genomic sequences were isolated from a carti- laginous shark species, Scyliorhinus torazame. The full-length open reading frame (ORF) of shark MT cDNA encoded 68 amino acids with a high cysteine content (29%). The genomic ORF sequence (932 bp) of shark MT isolated by polymerase chain reaction (PCR) comprised 3 exons with 2 interventing in- trons. Shark MT sequence shared many conserved features with other vertebrate MTs: overall amino acid identities of shark MT ranged from 47% to 57% with fish MTs, and 41% to 62% with mammalian MTs. However, in addition to these conserved characteristics, shark MT sequence exhibited some unique characteristics. It contained 4 extra amino acids (Lys-Ala-Gly-Arg) at the end of the b-domain, which have not been reported in any other vertebrate MTs. The last amino acid residue at the C-terminus was Ser, which also has not been reported in fish and mammalian MTs. The MT messenger RNA levels in shark liver and kidney, assessed by semiquantitative reverse transcriptase PCR and RNA blot hybridiza- tion, were significantly affected by experimental exposures to heavy metals (cadmium, copper, and zinc). Generally, the transcriptional activation of shark MT gene was dependent on the dose (0–10 mg/ kg body weight for injection and 0–20 lM for imme- rsion) and duration (1–10 days); zinc was a more potent inducer than copper and cadmium. Key words: metallothionein — tiger shark Scylio- rhinus torazame — heavy metals — gene expression Introduction Metallothioneins (MTs) are low molecular weight (6– 7 kDa) cytoplasmic heavy-metal-binding proteins. These cysteine-rich proteins have several biological functions in eukaryotic organisms, including (1) metal ion homeostasis, (2) detoxification of excess reactive heavy metal ions, and (3) providing a reserve of essential metals for other metalloproteins (Hamer, 1986; Muto et al., 1999). Owing to their highly inducible expression (transcriptional activation), MTs have also been used as molecular bioindicators to monitor the heavy metal pollution of aquatic or marine ecosystem, and to investigate the adaptive response of aquatic animals to metal-induced stres- ses (Hamilton and Mehrle, 1986; Roesijadi, 1994; Olsson, 1996; Langston et al., 2002). MT genes are known to be evolutionarily con- served in most vertebrates because of their key roles in a variety of enzymatic reactions (Olsson, 1993; Binz and Kagi, 1999). With interests in the molecular evolution of the MT genes among vertebrates numerous studies have been made on the structure and function of MT genes from fish, the evolution- arily lowest vertebrates. These include MT cDNA or genomic DNA genes from common carp (Hermesz et al., 2001; Chan et al., 2004), crucian carps (Ren et al., 2000), goldfish (Chan 1994), plaice (George et al., 1990), rainbow trout (Bonham et al., 1987), and ayu (Lin et al., 2004). Despite the numerous studies on fin-rayed bony fish MTs, there are few reports on the MT gene of cartilaginous fish species. The evolu- tionary position of these elasmobranch fishes rela- tive to other vertebrates including advanced bony fish and mammals makes them useful model sys- tems for studying the molecular evolution of verte- brate genes (Cho and Kim, 2002; Nam et al., 2002). Tiger shark (Scyliorhinus torazame) resides in the Correspondence to: Yoon Kwon Nam; E-mail: yoonknam@pknu .ac.kr 350 DOI: 10.1007/s10126-004-0043-y Volume 7, 350–362 (2005) ȑ Springer Science+Business Media, Inc. 2005
Transcript
Page 1: Shark (Scyliorhinus torazame) Metallothionein: cDNA ... · Shark (Scyliorhinus torazame) Metallothionein: cDNA Cloning, ... (sMT-1F, 5¢-ATGTCTGACACGAAGCCCTGTG- ... Bos taurus MT-I

Shark (Scyliorhinus torazame) Metallothionein: cDNA Cloning,Genomic Sequence, and Expression Analysis

Young Sun Cho,1 Buyl Nim Choi,1 En-Mi Ha,1 Ki Hong Kim,2 Sung Koo Kim,3

Dong Soo Kim,1 Yoon Kwon Nam1

1Department of Aquaculture, Pukyong National University, Busan 608-737, South Korea2Aquatic Life Medicine, Pukyong National University, Busan 608-737, South Korea3Biotechnology & Bioengineering, Pukyong National University, Busan 608-737, South Korea

Received: 22 April 2004 / Accepted: 7 October 2004 / Online publication: 4 June 2005

Abstract

Novel metallothionein (MT) complementary DNAand genomic sequences were isolated from a carti-laginous shark species, Scyliorhinus torazame. Thefull-length open reading frame (ORF) of shark MTcDNA encoded 68 amino acids with a high cysteinecontent (29%). The genomic ORF sequence (932 bp)of shark MT isolated by polymerase chain reaction(PCR) comprised 3 exons with 2 interventing in-trons. Shark MT sequence shared many conservedfeatures with other vertebrate MTs: overall aminoacid identities of shark MT ranged from 47% to 57%with fish MTs, and 41% to 62% with mammalianMTs. However, in addition to these conservedcharacteristics, shark MT sequence exhibited someunique characteristics. It contained 4 extra aminoacids (Lys-Ala-Gly-Arg) at the end of the b-domain,which have not been reported in any other vertebrateMTs. The last amino acid residue at the C-terminuswas Ser, which also has not been reported in fish andmammalian MTs. The MT messenger RNA levels inshark liver and kidney, assessed by semiquantitativereverse transcriptase PCR and RNA blot hybridiza-tion, were significantly affected by experimentalexposures to heavy metals (cadmium, copper, andzinc). Generally, the transcriptional activation ofshark MT gene was dependent on the dose (0–10 mg/kg body weight for injection and 0–20 lM for imme-rsion) and duration (1–10 days); zinc was a morepotent inducer than copper and cadmium.

Key words: metallothionein — tiger shark Scylio-rhinus torazame — heavy metals — gene expression

Introduction

Metallothioneins (MTs) are low molecular weight (6–7 kDa) cytoplasmic heavy-metal-binding proteins.These cysteine-rich proteins have several biologicalfunctions in eukaryotic organisms, including (1)metal ion homeostasis, (2) detoxification of excessreactive heavy metal ions, and (3) providing a reserveof essential metals for other metalloproteins (Hamer,1986; Muto et al., 1999). Owing to their highlyinducible expression (transcriptional activation),MTs have also been used as molecular bioindicatorsto monitor the heavy metal pollution of aquatic ormarine ecosystem, and to investigate the adaptiveresponse of aquatic animals to metal-induced stres-ses (Hamilton and Mehrle, 1986; Roesijadi, 1994;Olsson, 1996; Langston et al., 2002).

MT genes are known to be evolutionarily con-served in most vertebrates because of their key rolesin a variety of enzymatic reactions (Olsson, 1993;Binz and Kagi, 1999). With interests in the molecularevolution of the MT genes among vertebratesnumerous studies have been made on the structureand function of MT genes from fish, the evolution-arily lowest vertebrates. These include MT cDNA orgenomic DNA genes from common carp (Hermeszet al., 2001; Chan et al., 2004), crucian carps (Ren etal., 2000), goldfish (Chan 1994), plaice (George et al.,1990), rainbow trout (Bonham et al., 1987), and ayu(Lin et al., 2004). Despite the numerous studies onfin-rayed bony fish MTs, there are few reports on theMT gene of cartilaginous fish species. The evolu-tionary position of these elasmobranch fishes rela-tive to other vertebrates including advanced bonyfish and mammals makes them useful model sys-tems for studying the molecular evolution of verte-brate genes (Cho and Kim, 2002; Nam et al., 2002).Tiger shark (Scyliorhinus torazame) resides in the

Correspondence to: Yoon Kwon Nam; E-mail: [email protected]

350 DOI: 10.1007/s10126-004-0043-y � Volume 7, 350–362 (2005) � � Springer Science+Business Media, Inc. 2005

Page 2: Shark (Scyliorhinus torazame) Metallothionein: cDNA ... · Shark (Scyliorhinus torazame) Metallothionein: cDNA Cloning, ... (sMT-1F, 5¢-ATGTCTGACACGAAGCCCTGTG- ... Bos taurus MT-I

waters of East Asia including coastal areas of theKorean peninsula. Increasing pollution in coastalareas of South Korea caused by industrial activitiesgives rise to concern about the bioaccumulation oftoxicants such as heavy metals in marine inhabit-ants, of which this shark species is thought to be oneof main recipients. The objective of this study was toisolate and characterize the MT gene from tigershark, and to examine its transcriptional responsesto heavy metal exposure.

Materials and Methods

Fish Samples, RNA Isolation, and cDNA LibraryConstruction. Live shark specimens were purchasedfrom a local fish market and transferred to the lab-oratory, where tissue samples were surgically re-moved. Liver tissues from 3 males and 3 femaleswere pooled, and total RNAs were extracted usingthe TriPure Isolation Kit (Roche Molecular Bio-medical). From total RNA, the poly (A)+ RNAs werepurified using biotin-labeled oligo-d(T)20 and strep-tavidin-coated magnetic particles (Promega) accord-ing to the manufacturer’s instructions. Fivemicrograms of poly (A)+ RNA was used as templatefor cDNA synthesis. All the procedures for cDNAlibrary construction including cDNA synthesis,ligation, and packaging were performed followingthe protocol of k Zap cDNA Synthesis Kit (Strata-gene). The primary library (1.5 · 106 pfu/ml) wasamplified to 1.0 · 1011 pfu/ml, and an aliquot ofphage (5 · 107 pfu) was excised into plasmid pBlue-script SK vector in Escherichia. coli SOLR cells.

Isolation of Shark MT cDNA and Genomic ORFSequence. Of our expressed sequence tag (EST) clonesidentified in the liver of this species, SL0262 showedhigher similarity (e-value of 1E-71) with previouslyknown MT sequences (unpublished data) based on theBLASTx search against NCBI GenBank. A total of6144 random bacterial clones from the liver cDNAlibrary were arrayed on 4 nylon membranes (1536clones per membrane) and hybridized with the digo-xygenin-11-dUTP-labeled insert from EST cloneSL0262 prepared using the DIG DNA Labeling andDetection Kit (Roche Applied Biosciences). Hybrid-ization, washing, and signal detection were performedaccording to the manufacturer’s recommendations.The clones showing hybridization-positive signalswere selected and sequenced using an ABI 377 auto-matic sequencer (Applied Biosystems). The raw se-quence data collected were analyzed with thesequence editing software, Sequencher (Version 4.0;Gene Codes). The trimmed sequences were subjected

to similarity search against GenBank database (http://ncbi.nlm.nih.gov/BLAST).

Genomic open reading frame (ORF) sequence ofshark MT was isolated by PCR using 2 specificprimers (sMT-1F, 5¢-ATGTCTGACACGAAGCCCTGTG-3¢; and sMT-1R; 5¢-CTGAAACATCCAGTGTGTGG-3¢)designed based on the shark cDNA sequence.Genomic DNA was purified from the whole bloodusing the conventional sodium dodecyl sulfate(SDS) and proteinase K method (Nam et al., 2002).One microgram of genomic DNA was subjected toPCR containing 20 pmol of each primer and 0.5 UTaq DNA polymerase (Takara). Thermal cyclingcondition (30 cycles) was 94�C for 45 seconds, 58�Cfor 1 minute, and 72�C for 1 minute with an initialdenaturation step at 94�C for 3 minutes. Reactionvolume was 50 ll. The amplified product waspurified using spin column (Qiagen) and cloned intopGEM-T easy vector (Promega). The recombinantclones of correct size were selected, and the insertswere sequenced using ABI377 automatic sequencer(Applied Biosystems).

Phylogenetic Analysis of Shark MT. The aminoacid sequence deduced from the identified shark MT,cDNA was aligned with other MT sequences. Forphylogenetic analysis a total of 119 unique MT se-quences from teleosts (17 sequences for MT-A, 16 forMT-B, and 22 for unclassified MTs) and mammals(37 sequences for MT-I, 17 for MT-II, 7 for MT-III,and 3 for MT-IV) were obtained from GenBank(Table 1). Multiple alignment was carried out usingCLUSTAL W (Thompson et al., 1994). Gap openpenalty and gap extension penalty were set to 10 and0.05, respectively. The weight matrix was BLOSUM(for protein). The output alignment was manuallyedited using the GeneDoc program (http://www.psc.edu/biomed/genedoc/) for optimum alignment.Identities between shark MT and other MTs werealso calculated as percentages using the same pro-gram. The edited alignment was subjected to dis-tance and parsimony analyses to evaluate thephylogenetic relationship of shark MT with otherMT orthologues. Distance analysis was performedusing CLUSTAL W or PAUP* (Version 4.0b). Un-rooted phylogenetic trees were calculated using theneighbor-joining (NJ) method. Bootstrap replications(1000) were performed to obtain confidence esti-mates for each node in the tree. Distance was mea-sured by mean character difference. The maximumparsimony (MP) analysis was carried out usingPAUP*. Of 81 total characters (number of positionsin alignment including gaps), 40 characters wereparsimony-informative and gaps were treated asmissing. Heuristic search was performed based on

YOUNG SUN CHO ET AL.: EXPRESSION OF SHARK MT GENE 351

Page 3: Shark (Scyliorhinus torazame) Metallothionein: cDNA ... · Shark (Scyliorhinus torazame) Metallothionein: cDNA Cloning, ... (sMT-1F, 5¢-ATGTCTGACACGAAGCCCTGTG- ... Bos taurus MT-I

Tab

le1.

Met

allo

thio

nei

nP

rote

inSeq

uen

ces

use

din

Ph

ylo

gen

etic

An

alyse

s

Mam

mals

Fis

hes

Lab

el

Specie

sM

Tpro

tein

Access

ion

no.

Lab

el

Specie

sM

Tpro

tein

Access

ion

no.

M1-1

Hom

osa

pie

ns

MT

-IA

NP

_005937

Sh

ark

Scylo

rhin

us

ora

zam

eM

T(p

rese

nt

stu

dy)

AY

605090

M1-2

Hom

osa

pie

ns

MT

-IB

NP

_005938

A1

Ch

aen

oceph

alu

sacera

tus

MT

-AO

93593

M1-3

Hom

osa

pie

ns

MT

-IE

P04732

A2

Ch

ion

od

raco

ham

atu

sM

T-A

O13258

M1-4

Hom

osa

pie

ns

MT

-IF

NP

_005940

A3

Cypri

nod

on

sp.

MT

-AQ

92044

M1-5

Hom

osa

pie

ns

MT

-IG

NP

_005941

A4

Cypri

nu

scarp

ioM

T-A

O13269

M1-6

Hom

osa

pie

ns

MT

-IH

NP

_005942

A5

Dan

iore

rio

MT

-AN

P_5

71150

M1-7

Hom

osa

pie

ns

MT

-IJ

NP

_783321

A6

Noto

then

iacori

iceps

MT

-AP

62339

M1-8

Hom

osa

pie

ns

MT

-IK

isofo

rm1

P80296

A7

Para

ch

aen

ich

thys

ch

arc

oti

MT

-AO

93450

M1-9

Hom

osa

pie

ns

MT

-IK

isofo

rm2

AA

H28280

A8

On

corh

yn

ch

us

myk

iss

MT

-AP

09861

M1-1

0H

om

osa

pie

ns

MT

-IK

isofo

rm3

NP

_789846

A9

Salm

osa

lar

MT

-AC

AA

65929

M1-1

1H

om

osa

pie

ns

MT

-IL

P80297

A10

Salv

eli

nu

salp

inu

sM

T-A

AA

B66342

M1-1

2H

om

osa

pie

ns

MT

-IM

AA

L83902

A11

Sparu

sau

rata

MT

-AP

52727

M1-1

3H

om

osa

pie

ns

MT

-IQ

AA

O49186

A12

Th

erm

arc

es

Cerb

eru

sM

T-A

P52721

M1-1

4H

om

osa

pie

ns

MT

-IR

Q93083

A13

Tre

mato

mu

sb

ern

acch

iiM

T-A

O93609

M1-1

5H

om

osa

pie

ns

MT

-IS

AA

K26162

A14

Ch

ion

od

raco

rast

rosp

inosu

sM

T-A

CA

A09714

M1-1

6H

om

osa

pie

ns

MT

-IX

isofo

rm1

NP

_005943

A15

Gym

nod

raco

acu

ticeps

MT

-AC

AA

07555

M1-1

7H

om

osa

pie

ns

MT

-IX

isofo

rm2

AA

H18190

A16

Pagoth

en

iab

orc

hgre

vin

ki

MT

-AC

AA

07558

M1-1

8E

qu

us

cab

all

us

MT

-IA

isofo

rm1

P02800

A17

Cara

ssiu

scu

vie

riM

T-A

AA

N85819

M1-1

9E

qu

us

cab

all

us

MT

-IB

isofo

rm1

P02801

B1

Ch

aen

oceph

alu

sacera

tus

MT

-BP

52724

M1-2

0E

qu

us

cab

all

us

MT

-IA

isofo

rm2

SM

HO

1A

B2

Ch

ion

od

raco

ham

atu

sM

T-B

P62711

M1-2

1E

qu

us

cab

all

us

MT

-IB

isofo

rm2

SM

HO

BB

3C

hio

nod

raco

rast

rosp

inosu

sM

T-B

P62679

M1-2

2Su

ssc

rofa

MT

-IA

P49068

B4

Cypri

nu

scarp

ioM

T-B

Q9I9

I0M

1-2

3Su

ssc

rofa

MT

-IC

P79376

B5

Dan

iore

rio

MT

-BN

P_9

19249

M1-2

4Su

ssc

rofa

MT

-ID

P79377

B6

Dic

en

trarc

hu

sla

bra

xM

T-B

Q9P

TG

9M

1-2

5Su

ssc

rofa

MT

-IE

P79431

B7

Gym

nod

raco

acu

ticeps

MT

-BP

62713

M1-2

6Su

ssc

rofa

MT

-IF

P79378

B8

Moro

ne

saxati

lis

MT

-BP

62712

M1-2

7O

vis

ari

es

MT

-IA

S00808

B9

Noto

then

iacori

iceps

MT

-BP

62680

M1-2

8O

vis

ari

es

MT

-IB

P09577

B10

On

corh

yn

ch

us

myk

iss

MT

-BP

09862

M1-2

9O

vis

ari

es

MT

-IC

P09578

B11

Pagoth

en

iab

orc

hgre

vin

ki

MT

-BP

62681

M1-3

0B

os

tau

rus

MT

-Iis

ofo

rm1

P58280

B12

Para

ch

aen

ich

thys

ch

arc

oti

MT

-BP

62682

M1-3

1B

os

tau

rus

MT

-Iis

ofo

rm2

P55942

B13

Salm

osa

lar

MT

-BC

AA

65930

M1-3

2C

an

isfa

mil

iari

sM

T-I

O19000

B14

Salv

eli

nu

salp

inu

sM

T-B

AA

B66343

M1-3

3C

erc

opit

hecu

saeth

iops

MT

-IP

02797

B15

Tre

mato

mu

sb

ern

acch

iiM

T-B

P62678

M1-3

4C

ricetu

lus

gri

seu

sM

T-I

P02804

B16

Cara

ssiu

scu

vie

riM

T-B

AA

N85820

M1-3

5M

us

mu

scu

lus

MT

-IP

02802

C1

Barb

atu

lab

arb

atu

laM

TP

25128

M1-3

6O

rycto

lagu

scu

nic

ulu

sM

T-I

AA

A31147

C2

Cara

ssiu

sau

ratu

sM

TP

52723

M1-3

7R

att

us

norv

egic

us

MT

-IP

02803

C3

Cara

ssiu

sau

ratu

sM

TJC

2419

M2-1

Bos

tau

rus

MT

-II

isofo

rm1

P09579

C4

Eso

xlu

ciu

sM

TP

25127

M2-2

Bos

tau

rus

MT

-II

isofo

rm2

P55943

C5

Gad

us

morh

ua

MT

P51902

M2-3

Bos

tau

rus

MT

-II

isofo

rm3

SM

BO

2C

6G

ad

us

morh

ua

MT

CA

A65924

M2-4

Mu

sm

usc

ulu

sM

T-I

Iis

ofo

rm1

P02798

C7

Gob

iom

orp

hu

scoti

dia

nu

sM

TA

AO

89258

M2-5

Mu

sm

usc

ulu

sM

T-I

Iis

ofo

rm2

SM

MS2

C8

Icta

luru

spu

ncta

tus

MT

O93571

M2-6

Hom

osa

pie

ns

MT

-II

isofo

rm1

P02795

C9

Lit

hogn

ath

us

morm

yru

sM

TA

AL

37187

M2-7

Hom

osa

pie

ns

MT

-II

isofo

rm2

P80295

C10

Liz

aau

rata

MT

O13257

M2-8

Su

ssc

rofa

MT

-IIA

P79379

C11

Ore

och

rom

isau

reu

sM

TA

AP

14677

con

tin

ues

352 YOUNG SUN CHO ET AL.: EXPRESSION OF SHARK MT GENE

Page 4: Shark (Scyliorhinus torazame) Metallothionein: cDNA ... · Shark (Scyliorhinus torazame) Metallothionein: cDNA Cloning, ... (sMT-1F, 5¢-ATGTCTGACACGAAGCCCTGTG- ... Bos taurus MT-I

tree-bisection-reconnection (TBR) branch-swappingalgorithm. Bootstrap analyses were carried out usingboth fast stepwise-addition search (1000 replications)and full heuristic search (100 replications). Thebootstrap 50% majority-rule consensus trees fromboth NJ and MP analyses were visualized using theTreeView (Win32 1.52) program (http://taxon-omy.zoology.gla.ac.uk/rod/treeview).

Experimental Exposures to Heavy Metals. Toexamine transcriptional induction of the shark MTgene by heavy metal ions, 3 experimental exposuresto heavy metals were conducted. First, sharks (aver-age body weight, 320 ± 38 g) were given an intra-peritoneal injection of CdCl2 (Sigma) at different doselevels (2.5, 5.0, and 10.0 mg/kg body weight). A con-trol group injected with saline containing no cad-mium. The changes of MT mRNA levels in liver andkidney were monitored for 7 days. The injected fish(n = 16 per dose) were transferred to a 150-L well-aerated tanks and individuals (n = 4) were sampledfrom each group at 2, 4, and 7 days after injection.Second, the sharks were exposed to equal molarconcentrations of cadmium, copper, and zinc in orderto examine which heavy metal was the most potentinducer for shark MT. Fish were immersed in sea-water (150 L) containing 0, 5, 10, or 20 lM of eachheavy metal for 24 hours. The effect of extendeddurations (48 and 96 hours) on MT expression wasalso examined with a fixed dose (10 lM) of cadmium,copper, or zinc. Livers were sampled from 4 fishbelonging to each treatment group. Third, the timecourse of MT expression during the zinc exposurewas examined up to 10 days. Fish were immersed inseawater containing 0 or 10 lM of zinc, and 3 indi-viduals were randomly chosen from each tank at 1, 4,7, and 10 days after exposure. The starting level ofMT mRNA was also examined at day 0. Liver andkidney samples were subjected to RNA analysis.Water temperature was adjusted at 13� ± 1�Cthroughout the experiments.

RNA Blot Analysis. Total RNA was isolatedusing TriPure Isolation Kit (Roche Applied Bio-sciences) and treated with DNase I (10U/p lg totalRNA) for 30 minutes at 37�C in order to removepossible contaminating DNA. DNase I was inacti-vated by incubating the reaction at 90�C for 15minutes. One microgram of resulting total RNA wasspotted onto a positively charged nylon membrane(Roche Applied Biosciences) in a volume of 1 ll. Themembrane was processed according to the manu-facturer’s instructions and hybridized with digo-xygenin-11-dUTP-labeled full-length shark MTT

ab

le1.

Con

tin

ued

Mam

mals

Fis

hes

Lab

el

Specie

sM

Tpro

tein

Access

ion

no.

Lab

el

Specie

sM

Tpro

tein

Access

ion

no.

M2-9

Su

ssc

rofa

MT

-IIB

P79380

C12

Ore

och

rom

ism

oss

am

bic

us

MT

AA

P14678

M2-1

0C

an

isfa

mil

iari

sM

T-I

IQ

9X

ST

5C

13

Ore

och

rom

ism

oss

am

bic

us

MT

P52726

M2-1

1C

erc

opit

hecu

saeth

iops

MT

-II

P02796

C14

Ory

zia

sla

tipes

MT

AA

R30249

M2-1

2C

ricetu

lus

gri

seu

sM

T-I

IP

02799

C15

Pagru

sm

ajo

rM

TQ

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50

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3C

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lus

lon

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datu

sM

T-I

II4

8116

C16

Perc

afl

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52725

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sM

T-I

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17808

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coglo

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S00811

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spla

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aM

TS30567

M2-1

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MT

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CA

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MT

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80593

M3-2

Ovis

ari

es

MT

-III

AA

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MT

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tau

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MT

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M3-4

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MT

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P37360

M3-5

Hom

osa

pie

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MT

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P25713

M3-6

Mu

sm

usc

ulu

sM

T-I

IIP

28184

M3-7

Su

ssc

rofa

MT

-III

P55944

M4-1

Can

isfa

mil

iari

sM

T-I

VQ

9T

UI5

M4-2

Hom

osa

pie

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MT

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Mu

sm

usc

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47945

YOUNG SUN CHO ET AL.: EXPRESSION OF SHARK MT GENE 353

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cDNA. Labeling, hybridization, washing, and detec-tion were performed as described above. Thehybridized signal was analyzed using Quantity-Onesoftware (BioRad) to evaluate the relative intensityof the hybridized signal. Arbitrary values for inten-sity (INT/mm2) generated from the software wereused for the comparative analysis of hybridizationsignals among experimental groups. For Northernblot analysis, 10 lg of purified total RNA was sepa-rated by electrophoresis in a MOPS-formaldehydeagarose gel (1.2%). The RNA was transferred to anylon membrane using the capillary method (Sam-brook et al., 1989), processed according to the man-ufacturer’s instructions (Roche), and hybridized withDIG-labeled shark MT cDNA. The membranes (dotblot and Northern blot) were stripped and reprobedwith shark actin cDNA insert (EST clone; unpub-lished data) in order to normalize the MT mRNAlevels.

Semiquantitative RT-PCR Analysis. The differ-ential change of MT transcripts was examined withsemiquantitative reverse transcriptase PCR. Prior tosemiquantitative RT-PCR analysis, optimal condi-tions were established regarding the range of inputtotal RNAs (0.2–2 lg), the number of cycles (12–30cycles), and thermal cycling conditions for MT geneand actin gene (normalization control). The numbersof cycles were kept to a minimum and the RT-PCRswere linear in the range of input total RNA tested.As a negative control for each set of primers, RT-PCRs were performed in the absence of RT and RNA(data not shown). First-strand cDNA using Super-script II Reverse Transcriptase (Invitrogen) was gen-erated from 1 lg of total RNA (DNase-treated) witholigo(dT)18 primers. For PCR reactions, 0.5 U ExTaqDNA polymerase (Takara) and 2 ll cDNA were usedin 50 ll of amplification buffer containing 30 pmol ofprimers. The primer pair specific for shark MTcDNA was sMT-1F and sMT-1R as described above.The primer pair specific for shark b-actin was sACT1F 5¢-CTGTGCCCATCTAC GAAGGT-3¢ and sACT 1R 5¢-AGAGCGGTGATCTCCTT CTG-3¢. PCR was performedusing the iCycler (BioRad) under the following con-ditions: 94�C for 2 minutes (initial denaturation),94�C for 1 minute, 58�C for 1 minutes, and 72�C for1 minute. Numbers of cycles for MT and actin geneswere 25 and 20, respectively. PCR reactions wererepeated 3 times for each cDNA sample. Expectedsizes of PCR products of MT and actin are 280 and475 bp, respectively. PCR products were electro-phoretically separated on 2.0% agarose gels, and theethidium-bromide-stained bands were analyzed bydensitometry using Quantity-One software todetermine the relative mRNA levels.

Results and Discussion

Isolation and Characterization of Shark MT cDNAand Genomic Sequences. From 4 arrays, each con-taining 1536 randomly selected clones, 2 clonesshowed a positive signal with the MT probe in thefilter hybridization. Both clones contained full-length ORFs corresponding to shark MT mRNA, andhad the identical sequence composed of a 5¢-untranslated region (UTR) of 38 bp, a single ORF(204 bp) encoding 68 amino acids, and 3¢-UTR of 183bp excluding 71 bp of poly (A)+ tail. The consensussequence for polyadenylation was also found 19 bpupstream of the poly (A)+ tail (Figure 1, A). Thegenomic fragment isolated by PCR using sMT 1F and1R primers was 923 bp in length. It consisted of 3exons, 2 introns, and partial 3¢-UTR: exons I (34 bp),II (78 bp), and III (92 bp) were separated by introns I(203 bp) and II (449 bp). Consensus exon-intronboundary sequence (GT-AG) was clearly conserved(Figure 1, B).

Multiple Sequence Alignment Analysis. Theamino acid sequence of the putative shark MT de-duced from the cDNA sequences shared relativelyhigh similarity with other previously known MTsequences from vertebrates including mammals andbony fishes. Overall amino acid identities of sharkMT ranged from 41% to 62% with other MTs: aver-age identities were 57% ± 3% (range, 52%–62%) withmammalian MT-Is (37 sequences from 11 species),58% ± 2% (55%–61%) with mammalian MT-IIs (17sequences from 12 species), 47%–3% (41%–48%) withmammalian MT-IIIs (7 sequences from 7 species),50% ± 1% (50%–52%) with mammalian MT-IVs (3sequences from 3 species), 53% ± 2% (49%–56%)with teleost MT-As (17 sequences from 17 species),54% ± 2% (47%–55%) with teleost MT-Bs (16 se-quences from 16 species) and 52%–3% (47%–57%)with teleost MTs that had not been yet classified asMT-A or MT-B (tables not shown).

Optimized multiple alignment using shark MTand 119 orthologues generated 81 positions includ-ing gaps (only the selected sequences are shown inFigure 2). In mammalian MTs most MT-Is and IIscomprised 61 amino acids. Two exceptional MT-Isequences were human MT-IK (M1-8 in Figure 2)and horse MT-IA (M1-20 in Table 1; not shown inFigure 2), containing 62 and 60 amino acids,respectively. Human MT-IK (M1-8) had an excep-tional insertion of Ala at the 14th position, whichcannot be found in any other vertebrate MTs.Mammalian MT-IIIs consisted of 65 to 68 aminoacids with additional sequences in both b-domain(Thr or Ala at the 8th position) and a-domain (from

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the 64th to 71st positions): the consensus sequence(G/E)EGAEAE(A/E) and its modified forms inserted in a-domain were found in all of the previously knownmammalian MT-IIIs, but in no other MTs. Mam-malian MT-IVs had 62 amino acids with an insertionof Glu at the 8th position. Compared with themultiplicity of mammalian MTs, teleosts have beenknown to possess 2 distinct MTs; MT-A and MT-B.All the fish MT-Bs had 60 amino acids withoutvariation. Most, but not all, teleost MT-As alsocomprised 60 amino acids. Three salmonid MT-Asfrom Oncorhynchus mykiss (A-8 in Figure 2), Salmosalar (A-9), and Salvelinus alpinus (A-10) had anadditional Ala residue at the border between b-do-main and a-domain, which was not seen in MT-Bsfrom the same species. The insertion at the border

region has recently been reported in a nonsalmonidfish, ayu (Plecoglossus altivelis); however, the in-serted amino acid was Thr (C-17 in Table 1; Linet al., 2004).

Like many other MT sequences, shark MT con-tained a high proportion of Cys residues (20 [29.4%]of 68) as Cys-X-Cys or Cys-Cys forms. The positionsof most, but not all Cys residues in shark MT se-quences were well conserved when aligned to otherfish and mammalian sequences. In addition to theseconserved cysteine residues, shark MT shared sev-eral identical residues with other MTs. In b-domain,Asp (the 3rd position), Pro (the 6th position), and alsoLys (the 41st position) were conserved in all the fishand mammalian MTs. Serine at the beginning of a-domain (the 43rd position; except human MT-IB) andPro (49th position) were conserved in most verte-brate MTs. However, shark MT had some uniquefeatures, including 4 extra amino acids (Lys-Ala-Gly-Arg; 35th to 38th positions) which have not beenreported in any other vertebrate MTs. The positionof these additional amino acids was close to the endof b-domain (see Olsson, 1993). Analysis of genomicsequence indicated that these amino acids should beencoded by exon II of shark MT (see also Figure 1).However, the physiological function remains to bestudied. On the one hand, further comparisons ofshark MT with other teleost MTs based on thismultiple alignment showed that shark MT had un-ique insertions in b-domain which were missing inother MTs: Ser (the 2nd position), Thr-Lys (the 4thand 5th positions), and Pro (the 32nd position). Onthe other hand, there was a gap in shark MT at the15th position where many mammalian MTs had Glyor Asp, while most teleosts had Ser or Thr. SharkMT also had Gln at the 40th position (close to theend of b-domain), while most fish and mammalianMT-I, MT-II and MT-IIIs had Lys (mammalian MT-IV had Arg). Similarly, the amino acid at the 54thposition was Asn in shark MT, while most mam-malian and all the teleost MTs had Lys at this po-sition. The last amino acid residue at the C-terminusin shark MT was Ser, which has not been reported inany other vertebrate MTs: all the teleost MTs andmammalian MT-IIIs had Gln, while most mamma-lian MT-Is and MT-IIs showed Ala at the C-termi-nus.

Shark MT represented intermediate charactersbetween teleost and mammalian MTs, suggestingthat the shark MT might be an ancestral form ofvertebrate MTs. Shark MT shared homology withfish MTs, including the gaps at the 7th and 8thpositions in b-domain. Similarity between shark andteleost MTs was also found at the 72nd position: theLys residue was conserved in the majority of fish

Fig. 1. A: Nucleotide sequence of shark MT cDNA. Codingsequence is represented by uppercase type and noncodingsequence by lowercase. The deduced amino acid sequenceis indicated below the nucleotide sequence in single lettercode. The termination codon is indicated by an asterisk,and the putative polyadenylation signal (aataaa) is in bold-face. Sequences of primers sMT-1F and sMT-1R areunderlined. B: Genomic sequence of shark MT gene iso-lated by PCR using sMT-1F and sMT-1R primers (under-lined). Coding sequence is represented by uppercaseboldface. Exon-intron boundary sequences (gt-ag) are boxed.

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Fig. 2. Alignment of shark MT polypeptide sequences with mammalian MTs (M1-X for MT-I, M2-X for MT-II, M3-X forMT-III, and M4-X for MT-IV) and teleost MTs (A1 to A17 for MT-A and B1 to B16 for MT-B). Multiple alignment wasperformed with 120 unique sequences indicated in Table 1, but only representative sequences are shown here. For thespecies name and GenBank accession number of each sequence, see Table 1. Dots indicate the identities with shark MT.Hyphens represent gaps introduced for optimal alignment, and letters represent amino acids where substitutions occur.Conserved cysteine residues are boxed, and extra amino acids found only in shark MT are in boldface. The number ofamino acids is noted at the right of each sequence.

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MTs but was found in only 2 mammalian MT-IIIs. Inaddition to the similarity with teleost MTs, sharkshared homology with mammals in their MT se-quences. First, Arg at the 31st position was found inmany mammalian MT-Is and MT-IIs, but was innone of the fish MTs. Second, shark shared Arg-Serat 73rd-74th positions with most mammalian MT-Isand MT-IIs, but the consensus sequence at this po-sition in fish MTs was Thr-Cys. Third, the third Cysfrom the C-terminus (the 77th position) was con-served in shark and mammalian MTs, but wasmissing in all of the teleost MTs.

Construction of Phylogenetic Trees. The phy-logenetic relationships among 120 MT sequencesincluding shark MT inferred using NJ and MPmethods are shown in Figure 3 and Figure 4,respectively. In both NJ and MP analyses, sharkformed a unique branch. The NJ and MP analysesyielded similar phylogenetic hypotheses, with thesame nodes receiving bootstrap support higher than80% replicates in most cases. In the NJ tree the tel-eost MT, mammalian MT-IV, and mammalian MT-

III groups were supported by the bootstrap valueshigher than 90% replicates. However, mammalianMT-I and MT-II groups were not clearly divided fromeach other, and the large group consisting of bothMT-I and MT-II was characterized by 75% bootstrapsupport. Of a total of 16 highly supported nodes in NJtree (>90% of replicates), 8 nodes belonged to sub-groups of mammalian MT-I/II. Within the teleostgroup, Cypriniformes containing 10 MT sequenceswas characterized by 95% bootstrap support, 3 sal-monid MT-As (A8, A9, and A10) by 92%, and 2 iso-forms of G. morhua (C5 and C6) by 95%. Furtherinsights into teleost MTs have shown that the MT-Aand MT-B isoforms from the group containing Sal-moniformes (A8, A9, A10, B10, B13, and B14) appearto have originated before the species separations,because the clades within Salmoniformes are char-acterized by MT isoforms (MT-A or MT-B) ratherthan by species: e.g., arctic char MT-A (A10) is moreclosely related to rainbow trout MT-A (A8) than toarctic char MT-B (B14) (see also Bargelloni et al.,1999). A similar phenomenon is found in tilapiaspecies (see C11, C12, C13): Oreochromis mossam-

Fig. 3. Phylogenetic tree of MTinferred using NJ method. The 120unique MTs from fish andmammalian species (shown inTable 1) were used to construct thetree. The bootstrap values aspercentages shown at the nodes ofthe tree are based on 1000replications. Only values greaterthan 50% are shown. The GenBankaccession number of each sequenceis shown in Table 1.

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bicus MT isoform (C12) is more closely related toO. aureus MT (C11) than is O. mossambicus MTisoform (C13) (Figure 3).

The consensus tree summarizing results fromMP analysis is shown in Figure 4. The generaltopology of the MP tree was similar to that of the NJtree. The nodes receiving strong bootstrap support(‡80% in both fast stepwise-addition and full heu-ristic searches) characterized 14 clades. Clades Asare all isoforms of human MT-Is. Clades B and D are2 isoforms of horse (Equus caballus) MT-I andmouse MT-II, respectively. Clade C represents theMT-Is from 3 Muridae families. Clade G covers all ofmammalian MT-IIIs including 2 highly supportedclades, E and F. Clade H is a distinct group composedexclusively of previously known mammalian MT-IVs. The separation of MT-III and MT-IV groups athigh confidence levels is similar to the finding in NJanalysis. Unlike the NJ tree, the node for the teleostgroup is supported by relatively lower bootstrapvalues (68% in fast search and 73% in full search).Within this clade the formations of nodes are gen-erally in agreement with the expected taxonomicplacements. Fish species belonging to Cypriniformesformed a distinct clade I supported by 82% to 86%bootstrap values. Although the salmonid species(A8, A9, and A10) formed a distinct group in the NJtree, they did not receive high bootstrap support inMP analysis: only full heuristic search resulted in anode with 65% bootstrap support. Clade J represents2 isoforms of MT from G. morhua, and tilapia MTisoforms also formed a distinct clade K, as in NJanalysis (Figure 4).

Transcriptional Activation of Shark MT Geneby Intraperitoneal Injection of Cadmium. The con-centration of cadmium injected affected the induc-tion of shark MT transcripts. On the RNA blot assay,the basal level of MT expression was significantlyhigher in liver than in kidney. The MT mRNA levelof nonexposed fish was not significantly changed ineither tissue throughout the experiment. In kidney,all the dose levels (2.5–10 mg/kg body weight) of

Fig. 4. Consensus tree summarizing results from parsi-mony analyses using PAUP program under TBR algorithm(40 parsimony-informative sites; tree length, 302; consis-tency index, 0.5497; retention index, 0.8967; gaps treatedas missing). Numbers on branch nodes indicate bootstrapvalues (%) after 1000 replications using fast stepwise-addition search or 100 replications using full heuristic-search (in parentheses). Only values greater than 50% areshown. Uppercase labels (A to K) in circles represent dis-tinct clades supported by bootstrap values of 80% or higherin both searches. The GenBank accession number of eachsequence is shown in Table 1.

b

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cadmium injection revealed significant dose-depen-dent increases in MT transcripts at 2 days afterinjection. A transient increase in MT transcripts wasobserved up to day 4 after injection, which droppedto a lower level at day 7, although the level was still

elevated as compared to that in the control fish(Figure 5, A). The transient response of MT tran-script in kidney to heavy metal exposure has alreadybeen reported in rainbow trout (Norey et al., 1990).Liver also displayed a sharp increase of MT mRNA

Fig. 5. Levels of shark MT transcripts inkidney and liver induced by cadmiuminjection. A: RNA dot blot hybridizationprobed with shark MT cDNA. MT mRNAwas induced by intraperitoneal injection ofdifferent doses of cadmium (0–10 mg/kgbody weight). The MT mRNA levels wereassayed at 0, 2, 4, and 7 days after injection.B: Representative gel showing the RT-PCRproducts of the expected sizes (475 bp foractin and 280 bp for MT) fractionated in 2%agarose gel electrophoresis and stainedwith ethidium bromide. C: Densitometricanalysis (actin ratio) representing themeans of 3 independent PCRamplifications of a cDNA (see ‘‘Materialsand methods’’). Signal intensities (INT/mm2) of the bands were assigned by imageanalysis software (Quantity One). Standarddeviations are indicated by bars on thehistograms. Means with the same letterson each histogram within a day were notstatistically different at P < 0.05 based onanalysis of variance.

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levels by cadmium injection; however, the expres-sion pattern in liver was different from that in kid-ney. The maximum MT mRNA level was attainedin liver at day 4 and retained up to day 7, in contrastto the rapid drop of MT mRNA level at day 7 inkidney (Figure 5, A). A similar pattern of MTinduction at mRNA level was observed in semi-quantitative RT-PCR analysis (Figure 5, B and C).Actin gene showed steady-state expression withoutsignificant variations in kidney or liver tissues.However, MT transcripts were clearly induced bycadmium injection, and the increases in these tis-sues were dose-dependent, even though there wasnot a linear relationship between doses and mRNAlevels. As in RNA blot analysis, the induction of MTmRNA in kidney was transient; however, elevationwas retained in liver (Figure 5, B and C). The viewthat differential regulation of MT expression in dif-ferent organs might be due to the different rates ofmetal-ion uptake or excretion in the organs, whichcan also be affected by other metal-binding proteinsexisting in different organs, has been widely ac-cepted (see Gedamu and Zafarullah, 1993).

Induction of MT mRNA by Immersion Exposureto Cadmium, Copper, and Zinc. Sharks were ex-posed to equal molar concentrations (0, 5, 10, and 20lM) of 3 different heavy metal ions (Figure 6, A).Actin transcript was not changed during immersion,

but significant increases were detected in fish sub-jected to all the doses except nonexposed fish.According to the scanning densitometry, zinc wasthe more potent inducer than cadmium and copper:MT mRNA levels of fish exposed to zinc were al-ways higher than those of fish exposed to cadmiumand copper (pixel data not shown). The extendedexposures using a single dose (10 lM) also showedsimilar patterns of increase (Figure 6, B). The MTmRNA level induced by zinc was more than 2-foldthat of nonexposed fish at 48 hours after immersion.Cadmium induced slightly more MT mRNA in liverthan copper, but the difference was not significant.Further extension of duration up to 96 hours de-creased the difference in the induced mRNA levelsby the 3 heavy metals. Although the MT transcriptlevel induced by zinc was still slightly higher thanthose by cadmium and copper, the difference be-tween cadmium and copper was diminished at 96hours (Figure 6, B). Northern blot analysis using theRNA from the fish exposed for 96 hours showedsimilar results, but the difference was less comparedto that on RT-PCR (Figure 6, C). This result wassimilar to the findings of a previous report on rain-bow trout in which higher induction was detected byexposures to zinc and cadmium than to copper(Gedamu and Zafarullah, 1993). Olsvik et al. (2001)also reported that brown trout from a stream con-taminated with cadmium and zinc showed signifi-

Fig. 6. Semiquantitative RT-PCR analysisof RNA from fish exposed to cadmium,copper, and zinc. A: Induction of hepaticMT mRNA by immersing sharks in equalmolar concentrations (5, 10, or 20 lM) of 3heavy metals for 24 hours. C0 indicates thestarting level of MT transcripts assayed atday 0. C1 is the nonexposed controlassayed at 24 hours after immersion(immersion in water containing no heavymetal). B: MT mRNA levels induced byexposures to heavy metals at 10 lM for 48and 96 hours. C0 is the starting level of MTtranscripts and C1 lanes are nonexposedcontrols assayed at 48 and 96 hours afterimmersion. C: Northern blot hybridizationto show MT mRNA levels in shark liverinduced by exposure to cadmium, zinc,or copper at 10 lM for 96 hours. Tenmicrograms of liver total RNA wastransferred to nylon membrane and probedwith digoxygenin-labeled full-length sharkMT cDNA probe. Control hybridizationwas a with shark b-actin cDNA fragment.

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cantly higher MT level than fish from a copper-pol-luted river. The differential response of the MT geneto different kinds of metal inducers might be due tonot only the differential affinity of the heavy metalsfor a metal-binding transcription factor but also thedifferent rates of metal flux and availability (Ged-amu and Zafarullah, 1993). However, Boeck et al.(2003) proposed that there also might be significantvariations among fish species in the regulatorycapacity for a specific metal homeostasis: for exam-ple, cyprinid fish species exhibited higher tolerancefor copper exposure and showed much more positivecorrelation between tissue copper levels and tissueMT levels than salmonid fish.

Using the most potent inducer, zinc, the timecourse of MT expression was monitored up to 10days (Figure 7). In both kidney and liver, significantincrease of MT transcripts was detectable at day 1 bysemiquantitative RT-PCR. Although there was atrend toward higher induction with longer durationof zinc exposure, the level of MT transcripts rapidlyreached its maximum at day 4 (kidney) and at day 7(liver). Unlike the transient pattern of expression inkidney by cadmium injection, however, there was noevidence that the MT mRNA dropped rapidly afterreaching maximum induced level: induced MTtranscripts remained elevated up to 10 days withoutany significant decrease. Positive correlations be-tween MT levels and periods of exposure to heavymetals have also been observed in other teleostspecies at mRNA or protein levels (George et al.,1996; Boeck et al., 2003; Lin et al., 2004). The shortperiod required to reach maximum induction ob-served in this study might be due to the relativelyhigh concentration of doses (10 lM). To consider theenvironmentally realistic doses, further experimentsshould be conducted using lower concentrations andmore extended periods. It also would be valuable toexamine the expressed profile of other stress-res-ponsive genes together with MT transcripts during

heavy metal exposures, because stress factors otherthan metal ions also could affect MT mRNA levels.

In summary, a novel MT cDNA and genomicsequence was isolated from a cartilaginous sharkspecies, Scyliorhinus torazame. The shark MTshared homologous traits with MTs from both ad-vanced bony fishes and mammalian species, sug-gesting that the shark MT may be the ancestral formof vertebrate MTs. However, it is not clearly under-stood yet whether or not the present type of sharkMT gene belongs to subtype MT-A or MT-B. Furtherexperiments on the genomic structure of shark MTgene including genomic Southern blot hybridizationand inverse PCR will be useful for better under-standing the origin of ancestral MT gene of verte-brates. The highly inducible expression of shark MTgene by various metal ions may also allow the use ofMT transcripts as molecular biomarkers to addressenvironmental contamination by heavy metals.

Acknowledgments

This study was supported by Korea Sea Grant Pro-gram from Ministry of Maritime Affairs and Fisheries.

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362 YOUNG SUN CHO ET AL.: EXPRESSION OF SHARK MT GENE


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