クサガメからの完全長dmrt1 cDNAのクローニング

誌名誌名 The journal of veterinary medical science

ISSNISSN 09167250

著者著者星, 英之中尾, 亜矢

巻/号巻/号 70巻7号

掲載ページ掲載ページ p. 687-692

発行年月発行年月 2008年7月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

FULL PAPER Toxicology

Molecular Cloning of Full-Length Dmrt1 cDNA of Reeves Turtle (Chinemys reevesii)

Hidenobu HOSHII)* and Aya NAKAOI)

J)Department 01 Veterinary Public Health， Division 01 Veterinary Science， Graduate School 01 Life and Environmental Sciences， Osaka Pr，φcture Universiの，1-1 Gakuen-cho， Naka-ku， Sakai， Osaka 599-8531， Japan

(Rec巴ived6 December 2007/Accepted 3 April 2008)

ABSTRACT. We cloned and sequenced a full-Iength open reading frame turtle dmrt1 cDNA (Crdmrtl) that was 1，504 bp in length and encoded 371 amino acid residues. RT-PCR analysis in di百erenttissues of adult male turtle showed that出eCrdmrt1 cDNA fragment was only det閃 tedin the testis. The amino acid sequence derived from Crdmrt1 demonstrated high homology to sequences from dmrtl of Pelodiscus sinensis (92% identities and 93% positives) and Elaphe quadrivirgata (75% identities and 83% positives). The deduced amino acid from Crdmrt1 contained a conserved DM domain， a male-specific motif， and a P/S-rich region. In DMRTl from reptiles，

birds， mammals，創nphibians，and fish， the amino acid identities and positives for DM domains were 85-100% and 88-100%， respec-tively， those for male-specific motifs were 47-100% and 60--100%， and those for P/S-rich regions were 24-100% and 35-100%. The module consisted of intertwined CCHC and HCCC Zn2+-binding sites in the DM domain and was conserved in all 11 species analyzed in this study. Amino acid sequences of Crdmrtl and previously reported DMRTls， DMRT2s， and DMRT3s were su同巴ctedto phylo-genetic analysis. The resultir沼田巴 showedthat CrDMRTl belongs to DMRTl， and turtles are a sister group to a cluster of birds and snakes. This is出eflfst study of the cloning of full-Iength dmrt1 from a reptil巴.

KEY WORDS: cloning， dmrt1， full-Iength cDNA， phylogenetic analysis， turtle.

Members of the dmrt (doublesex-and mab-3・related住組-

scription factor) gene family have been isolated indep巴n-

dently in both vertebrates and invertebrates， and are of special interest because of their rol巴sin sexual differentia-

tion [25]. A dmrtl was originally assigned to band 9p24.3， which is the critical region for sex reversal in humans [19].

The DMRT1 protein contains a zinc finger-like DNA-bind-

ing motif called the DM domain， which is shar巴dbetw巴en

Drosophila doublesex (dsx) [4] and Caenorhabditis elegans

Male ABnormal (mab-3) [21]. Both dsx and mab-3 play a key role in sex determination in these species.

A dmrtl is considered to be one of the essential genes

involved in the testicular differentiation cascade in mam-

mals， birds， reptiles日5]，amphibians [22]， and fish [14]. In

many reptiles， sex is determined by the temperature at which the eggs are incubated [3， 7]. In alligators (Alligator missis・sippiensis)， dmrtl expression was initially det怠ct疋dusing RT-PCR in the urogenital systems of embryos incubated at

both male-and female-producing temperatures; how巴V巴r，

gonadal expression subsequently became higher in develop-

ing male embryos th組 infemale embryos [23]. In Trache-mys scripta， up-regulation of dmrtl was observed in the developing male gonads of a turtle incubated at male-repro-

ducing temperature [16]. Chinemys reevesii is the turtle

commonly seen in lakes， rivers， and ponds in Japan， China，

Korea， and Taiwan. C. reevesii also has temperature-depen-dent sex determination [11]， however， it is not clear whether dmrtl is contributed in gonadal sex-differentiation in出is

turtle. For出巴 flfststep， we decide to clone dmrtl cDNA in

* CORRESPONDENCE TO: HOSHI， H.， Dep紅白nentof Veterinary Public Health， Division of Veterinary Science， Graduate School of Life and Environmental Sciences， Osaka Prefecture University， 1-1 Gakuen-cho， Naka-ku， S紘ai，Osaka 599-8531， Japan. e-mail: hhoshi@vet.osakafu-u.ac.jp

J. Vet. Med. Sci. 70(7): 687--692， 2008

C. reevesii. In reptiles， there is no report on the cloning offull-Iength

open reading frame (FL-ORF) dmrtl cDNA. In出isstudy， we succ巴ssfullyisolated FL-ORF dmrtl cDNA in C.

reevesii (Crdmrtl) and compared its d巴ducedamino acid

sequence with those of dmrtl genes reported in a database.

We also determined the expression of Crdmrtl in several

tissues of adult male turtle. To our knowledge， this is the first study on the cloning of FL-ORF cDNA of dmrtl from

a reptile.

MATERIALS AND METHODS

Aminals: All experiments were conducted in accordance

with the Guidelines for Animal Welfare in Research and

Education of Osaka Prefecture University. Three male C. reevesii turtles (mean carapac巴length士S.D.= 128 :t 6 mm; mean weight :t S.D. = 410 :t 24 g) were purchased from

Shimizu Laboratory Supplies， Kyoto， Japan. The turtles

were fed commercial di巴t(Tetra ReptoMin， Tetra Japan， Tokyo， Japan) and kept at 26 :t 40C on a 12.hr: 12・hr

light:dark cycle. Cloning of C. reevesii cDNA encoding dmrtl: Testes

samples were collected from an adult male turtle under ket-

amine anesthesia. The samples were quickly frozen in liq-

uid nitrogen， stored at -80oC and were subsequ巴ntlyused

for RNA isolation. RNA was extracted using TRlzol

reagent (Lif巴 Technologies，Gaithersburg， U.S.A.).

Poly(A)+ RNA was purified using Poly(A) Tract System

(Promega， Madison， U.S.A.) according to the manufac-

turer' s instructions. The t巴sticularpoly(A)+ RNA (500 ng)

was reverse-transcribed using random hexamer as a primer and SuperScript 11 r巴versetranscriptase (Invitrogen， Car-

688 H. HOSHI AND A. NAKAO

land， U.S.A.) according to the following schedu¥e: 1 cycle of 10 min at 250C， 50 min at 420C， and 15 min at 70oC. The PCR was performed in 50μ.1 containing 0.5μl ofcDNA， 5 μl of 10 x Gene Taq Universal Buffer， 4μl of 2.5 mM dNTP mix， 2.5 U of Gene Taq NT polymerase (Nippon Gene，

Tokyo， Japan)， and 50 pmol of each primer (DMRT5¥5'-GGA CAC AAG CGG TTC TGC ATG-3'; DMRT3'，デーGTA TGA GTG CAT CCG GTA CTG-3')， designed from the dmrtl cDNA sequence of Trachemys scripta. The tem-perature conditions for the PCR were as follows: one cycle of denaturation at 940C for 2 min， 35 cycles of denaturation at 940C for 1 min， annealing at 500C for 1 min， and exten-sion at 720C for 1 min， and fina1ly one cycle of extension at 720C for 5 min. For rapid amplification of cDNA ends (RACE)，出eforward and reverse primers (DMRT/GSP1，

5' -CCT GAA TGA GCA TCC GTC CCT CTG ATG GGG-3'; DMRT/GSP2，デーGCGCAG GTG GCA TTG AGA AGG CAG CAG GCC-3') were d巴sign巴dfrom the partial cDNA sequence of C. reevesii dmrtl. The 5'-end and 3'-end sequences of the dmrtl cDNA were determined by RACE-PCR using the Marathon ™ cDNA Amplification Ki~ (Clonetech Laboratories， Pa10 Alto， U.S.A.). The tempera-ture conditions for デー and3' -RACE were as follows: fi ve cycles of denaturation at 940C for 5 sec and annealing at 720C for 4 min，日vecycles of denaturation at 940C for 5 sec and annealing at 700C for 4 min， and 25 cycles of denatur-ation at 940C for 5 sec and annealing at 680C for 4 min. PCR products with more than one band were separately iso-lated and cloned into th巴pCR4-TOPO plasmid vector using TOPO TA Cloning Kit (Invitrogen). One Shot TOPlO E. coli cells (TOPO T A Cloning Kit) were also transformed into出evector.

Plasmid DNA was isolated from the cloned E. coli using a QIAprep Spin Miniprep Kit (Qiagen).

The nucleotide s巴quencesof the cDNA clones were determined using a Long-Read Tower DNA Sequencer (Amersham Pharmacia Biotech， Tokyo， Japan) after per-forming the sequencing reaction with the Thermo Sequence Cy5 and Cy5.5 Dye Terminator kits (Amersham)

Sequence alignment and phylogenetic analysis: Multiple alignment of arnino acid sequences was performed using MAFFT version 6 [13]. Aligned amino acid sequences were used to construct a phylogenetic tree by the neighbor-join-ing method using NJprot [18]. Bootstrap analysis with 1，000 replicates was used to assess the strength of出巴 nod白

血血e紅白 [8].Accession numbers of the sequences are as follows:

Crdmrtl (this study)， AB365876; Gallus dmr.

Analysis of dmrtl expression in the organs of the adult male turtles: For RT-PCR analysis， testes， livers， spleens，

brains， and kidneys from the adult male turtles were removed under anesthesia， quickly froz巴nin liquid nitrogen and stored at -80oC until further processing. Total RNA was isolated from the tissues of the turtl巴susing TRIzol reagent (Life Technologies) and was treated with RQl RNase-Free DNase (Promega). The DNase-treated RNA samples were stored at -80oC until the next procedure.

The cDNA was prepared using 1μg of DNase-treated RNA and was reverse-transcribed with ReverTra Ace reverse transcriptase (Toyobo， Osaka， Japan) and oligo(dT) 20 primer according to出emanufacturer's protocol. PCR was performed using 50μl containing 0.5μlcDNA，5μlof 10 x Gene Taq Universal Buffer， 4μl of 2.5 mM dNTP mix， 2.5 U Gene Taq NT polymerase (Nippon Gene)， and 50 pmol of each primer (CrDM5RT，デー CGCAGG TGG CAT TGA GAA GGC AGC -3'; CrDM3RT，ター TCTGCCATTGGT TTC CTG A TI GGC・3')designed from Crdmrt1. Theβactin was used as an internal control. The primers for βactin (Actin5RT， 5' -TGT GAT GGT GGG AA T GGG TCA G -3'; Actin3RT，デー GGATCT TCA TGA GGT AGT CCG -3') were designed form the sequence of snapping tur-tleβactin (Chelydra serpentina， GenBank accession No. AF541916). The amplification of cDNA was done in one cycle of denaturation at 940C for 2 min， 35 cycles of dena-turation at 940C for 1 min， annealing at 550C for 1 min， and extension at 720C for 1 min. The RT-PCR products were separated by巴lectrophoresison a 2% agarose gel and visu-alized by ethidium bromid巴staining.

RESULTS

Ap紅 tialsequence of C. reevesii dmrtl cDNA was ampli-fied by RT-PCR using the DMRT5' and DMRT3' primers， yielding a 615-bp fragment. W巴designedthe DMRT/GSP2 and DMRT/GSPl primers from the sequence of 615-bp fragment for 3' -and 5' -RACE， respectively. The FL-ORF Crdmrtl cDNA (AB365876) isolated from the testicular adaptor ligated double strands cDNA consisted of 1，116 bp of ORF， 181 bp of デーUTR，and 207 bp of 3' -UTR. The Crdmrtl cDNA encod巴d371 amino acids with a predicted molecular mass of 40 kDa

The arnino acid sequence d巴rivedfrom Crdmrtl demon-strated high homology to the sequences from dmrtl s of Pel-odiscus sinensis (92% identities and 93% positives)， Elaphe quadrivirgata (75% identities and 83% positives)， Trim-eresurusflavoviridis (75% identities and 82% positives) and Sus sc問向 (69%identities and 79% positives).

The predicted amino acid sequence of Crdmrtl had a conserved DM domain， a male-specific motif， and a P/S-rich region (Table 1). The alignrnent of the DM domain from 11 species showed 85-100% identities and 88-100% positives to出eDM domain from Crdmrtl (Table 1 and Fig lA). Cysteines and histidines indicated as site 1 and site II (Fig. lA) were also conserved in 11 species. The alignment of amino acid sequences of出ema¥e-specific motif showed

TURTLE DMRTl 689

Table 1. Amino acid identities (%) and positives (%) among aligned vertebrate DMRTl sequences

Species DM domain Mala-specific motif P/S-rich motif

ldentities Positives ldentities Positives ldentities Positives

Pelodiscus') 100 100 87 87 100 100 Elaphea) 100 100 80 87 85 95 Gallusa) 97 97 93 93 89 89 Homo 97 97 100 100 85 93 Sus 97 97 93 1∞ 89 93 凡1us 98 98 80 87 93 96

Pleurodeles 97 98 80 87 89 93 Rana 92 97 86 86 81 85

Xenopus 97 98 80 87 81 85 Epinephelus 89 91 73 80 33 40 Takifugu 85 88 47 60 24 35

Percentage id巴ntitiesand positives were calculated from comparisons of amino acid s叫uencesof each species to the sequence of CrDMRT 1. Gaps were excluded for the alignment calculations. Accession numbers of DMRT1s used are listed in MATERIALS AND METHODS. a)The C-or N-terminal sequences of the Pelodiscus， Elaphe， and Gallus DMRTl proteins are not contained in the database.

47-100% identities and 60-100% positives in all species (Table 1 and Fig. 1B). The alignment of amino acid sequences of白巴 P/S・richregion showed 81-100% identi-ties and 85-100% positives within the birds， reptiles， mam-ma1s， and amphibians. In fish; however， homology ofthe P/ S-rich region was low (Table 1 and Fig. 1 C)

The phylogenetic trees generated from the alignment of amino acid sequences of DMRT1， DMRT2， and DMRT3 showed that CrDMRT1 belonged to DMRT1 (Fig. 2). CRDMRT1 and Pelodiscus DMRT1 constituted a sister group of Gallus DMRT1 and Elaphe DMRTl.

The expression of Crdmrtl was examin巴dby RT-PCR in five tissues taken from白巴 adultma1e知rtles.The Crdmrtl fragment (477 bp) was observed on1y in the testis，加dit was not possible to amplify this仕agmentfrom any other adult ma1e tissues出osewere tested (Fig. 3). As expect巴d，出eβ actin fragments (452 bp) were detected in a11 five tissues.

DISCUSSION

We cloned FL-ORF dmrtl cDNA from the turtle Chine-mys reevesii. To our knowledge， this is the first study of the isolation of FL-ORF dmrtl cDNA from a reptile.

Partial ORF dmrtl cDNA sequences of reptiles were reported from Pelodiscus sinensis， Elaphe quadrivirgata， Trachemys scripta (AF201387組 dA Y316537)， Trimeresu-rus flavoviridis (AB254801)， Calotes versicolor (AF464141) and Aspidoscelis inornata (EF564796). The

deduced amino acid sequence of CrDMRTl shows high homology to DMRT1 in白巴 reptilesmentioned above.

DMRTl has been reported to have three conserved regions: a DM domain， a male-specific motif， and a P/S-rich region [10， 20]. As expected， in the present study出巴 DMdomain of CrDMRT1 showed a very high sirnilarity to that of Pelodiscus and DMRT1s in other v巴rtebrates(> 85% identities). Particularly， site 1 (CCHC) and site II (HCCC) in the DM domain were conserved in a1111 species ana1yzed in this study. These residues participate in two intertwined

Zn2+ーbindingsites [27]. The mutagenesis test in the DM domain of dsx in Drosophila demonstrated白紙 theseeight motiιspecific histidines and cysteines that are engaged in zinc coordination are a11 critical and紅巳 notinterchangeable [26].

Another conserved region， the male-specific motif， in CrDMRT1 shows very high similarity to DMRT1s from Homo sapiens and other vertebrates (> 73% identiti巴s)except for Takifugu rubripes (47% identities). In Droso-phila melanogaster， two DM-domain genes， male-specific dsX" and female-specific ds:x!， have been reported [1]. They conserve the DM-domain， but on1y dsX" has a male-specific motif near the C-terminus [1， 24]. In vertebrates， DM-domain cDNAs were isolated from tilapia testis and ovary [10]. Despite high homology in the DM domain， a ma1e-specific motif was absent in DM domain cDNA isolated from the ovary.

P/S-rich regions， rich in proline and serine residues，紅巳

found at corresponding positions in th巴 C-terrnInalpart of all D恥1RT1proteins， as reported by Brunner et al. [2]. Homology within the P/S-rich region is high (>81 % identi-ties) throughout the vertebrates巴xceptfish (24% and 33% identities). Less conservation in the P/S-rich region than in the DM domain was reported in fish [9]. Until the sequence of Crdmrtl was reported， dmrtl in T. scr伊ta(A Y316537) was the only record that indicated the sequence of a com-plete P/S-rich region in reptiles， because the sequences of dmrtl s in Pelodiscus and Elaphe were partial. The arnino

acid sequence of the P/S-rich motif in T. scripta dmrtl

shows 96% identities to Crdmrtl. This result indicates that DMRT1s in reptiles have a conserved P/S-rich motif.

Phylogenetic analysis of DMRT1s， DMRT2s， and DMRT3s showed出atCrDMRT1 belongs to DMRTl. As expected， we found出at出esister taxon of CrDMRTl was Pelodiscus DMRTl. The phylogenetic position of turtles is a controversial issue. A recent phylogenetic analysis showed出atturtles are a sister group to a monophyletic clus-ter of crocodiles and birds [12]. Previously reported

690 H. HOSHI AND A. NAKAO

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Ch inemys 59 : Pe/odiscus 56 E/aphe 57 Ga/ /us 6 Homo 68・

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Fig. 1. Multiple alignments of CrDMRT1 and DMRT1s of other v巴rtebrates.(A) Alignm巴ntsof DM domains in DMRTls of verte-brat巴s.Cysteines and histidines that coordinate Zn2

+ are aligned as two intertwined binding sites (arrows): site 1 and site II. (B) Align-ments of the male-specific motif in DMRTls of vertebrates. (C) Alignments of P/S rich region in DMRT1s of vertebrates. ldentical amino acids are indicated as white letters in black boxes， and positive amino acids are indicated as black letters in gray boxes. Gen-Ba凶(TMaccession numbers ofDMRTls used are listed in MATERIALS AND METHODS

sequences of DMRT1s in crocodil巴swere too short for the

phylogenetic analysis of出1Sstudy; however， the sequences of DMRTls in turtles show that turtles are a sister group to

a cluster of birds. This r巴sultsupports the hypothesis of

Iwabe et al. [12]. In the present study， RT-PCR using a Crdmrtl-specific

primer pair was us巴dto analyze出巴 expressionof dmrtl in various tissues of the adult male turtles. Sirnilar to the situ-

ation in the human [5]， mouse [6， 20]， rainbow trout [14]， Rana rugosa [22]， and Xenopus laevis [17]， a Crdmrtl cDNA fragment was detect巴donly in testis among th巴 tis-

sues tested.

In conclusion， we isolated a FL-ORF dmrtl cDNA

(Crdmrtl) from Chinemys reevesii. Crdmrtl has three con-

served regions: a DM domain， a male-specific motif， and a

P/S-rich region. A phylogenetic analysis of Crdmrtl indi-cates that turtl巴sare phylogenetically closely related to birds

and snak巴s.

ACKNOWLEDGMENT. This work was supported by

Grants-in-Aid for General Scientific Research from the

Ministry of Education， Culture， Sports， Science and Tech-nology (Hoshi: 14042257) of Japan.

TURTLE DMRTl 691

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DMRT1

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Fig. 2. Phylogenetic analysis of DMRTls， DMRT2s， and DMRT3s. The tree， based on a1ignments gen-erated using MAFFT version 6 (L-町S-imode， a11 ungapped sites， 101 amino acids， boots回 pneighbor-joining me出od，l，∞opseudosamples)， is rooted. Values indicated on出ebranches are boots回 pvalues (%). GenBank ™ accession numbers of DMRTl s， DMRT2s and DMRT3s used are indicated in MA TE-RIALS AND METHODS.

T L S B K

dmrtJ 477 bp

4メタ-actin452 bp

Fig. 3. Representative RT-PCR analysis of CrdmrtJ andβactin mRNA expression in various tissues of an adult male turtle. T， testis; L， 1iver; S， spleen; B， brain; K， kidney.

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