Sox. Sox. Sequencing of Sox gene HM G2box and intron genetic variability of Sox9 gene in conser2 vative HMG2box in t wo f ishes 3

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1 50 (4) : A cta Zoologica S inica Sox Sox Sequencing of Sox gene HM G2box and intron genetic variability of Sox9 gene in conser2 vative HMG2box in t wo f ishes 3 CHEN Jin2Ping 1 2 SUN Da2Jiang 2 L IAN G Bing 1 SHI Xing2Di 3 WU Wen2Hua 2 ZHAN G Shu2Yi Institute of Zoology The Chinese Academy of Sciences Beijing China 21Engeering and Technology Center of Sturgeon Breeding and Cultivation Chinese Academy of Fishery Sciences Beijing China 31 College of Life Science Beijing Normal University Beijing China Abstract Four different Sox genes in Amur sturgeon Acipenser schrenckii and five Sox genes in chum salmon On2 corhynchus keta were identified for the first time in this paper and there was no sexual difference for these genes in these two fishes. For Amur sturgeon the encoded amino acid sequences exhibited 98 % 98 % 98 % 86 % of sequence homol2 ogy with those reported for human S OX HM G2box respectively and for chum salmon the encoded amino acid se2 quences exhibited 93 % 98 % 98 % 98 % 96 % of sequence homology with those reported for human S OX HM G2 box respectively. Two different forms of Sox9 gene in chum salmon were found and Sox9 contained a 303 bp and 258 bp introns in the HM G box for chum salmon respectively while 572 bp for Amur sturgeon. No clear similarity was ob2 served between the introns in the Sox9 HM G box of different fishes but the introns occur in the HM G box region be2 tween the second and third bases of the codon for arginine at residue 42 in HM G box for the three fishes and obey the rule of GT2A G. and the encoded amino acid sequences in the three Sox9 genes HM G2box are very conservative in the two fishes (100 % identification). Identification of these genes is a potential step in understanding development regulations including sex determination in Amur sturgeon [ Acta Zoologica Sinica 50 (4) : ]. Key words Amur sturgeon Acipenser schrenckii Chum salmon Oncorhynchus keta Sox gene HM G2box Exon Intron Genetic variability Sox HM G Sox HM G ( HM G2box) ( Pevny et al. 1997) ( Foster et al. 32 Sox 1994 ; Pevny and Lovell2Badge 1997 ; Schepers et al ; Clarkson and Harley 2002 ; Chang et al ; Montero et al ; Galay2Burgos et (Bowles et al ; Nagai 2001 ; Chang et al ; Lunde et al. 2004) Sox9 HM G 79 al. 2004) AMH ( KSCX22SW2118) ( No ) [ This re2 search was funded by the Innovative grants of Chinese Academy of Sciences (No. KSCX22SW2118) and the National Nature Science Founda2 tion of China (No ) ] 33 (Corresponding author). E2mail : ν 2003 Acta Zoologica Sinica

2 DNA (Opening reading frame ORF) p GEM2T ( Promega ORF ) 5 (cdna 15 ( A ci penser schrenckii ) ( O n2 ) DNA corhynchus keta) Sox9 HM G Sox9 HM G Sox9 DNA club 4 Sox ( g 95 % ; Sox RT2PCR D NA RNA DNA / A Y A Y627287) Yue and Orban ( 2001) Sambrook et al. (1999 ) TE ( p H 810 ) - 20 RNA Trizol ( Invit2 rogen ) 113 PCR RT2PCR Sox9 HM G2box A S Sox11 1C 2 A S2 Sox9U 5 2CC ( CGT) CA ( CT) GT ( GC) AA ( A G) A GACC ( A GC ) A T23 ; Sox9L 5 2CC (GT) (CT) C ( GT) ( GCT) GGCTG (A G) TA (CT) TT (A G) TA23 ( Takamatsu et al ; Liu et al ; Chiang et al ; Yan et al ; Yokoi et al. 2002) Sox9 HM G2 box 76 PCR PE9700 PCR 6 5 Sox 25 l : 94 5 min s s 72 1 min A Y A Y A Y A Y581213) 10 min RT2PCR RNA (1 228 bp : CS Sox8 CS Sox10 g) SuperScript ( Invit2 rogen ) cdna cdna Sox9U Sox9L PCR PCR Sox ) ; Genbank Sox cd2 NA ( : A Y A Y http : / / ncbi1nlm1nih1gov/ BLAST/ ( 76 ) Sox9 98 % Sox9 100 % A S Sox9 Sox 98 % 98 % 86 % A S Sox2 A S Sox4 Sox2 A S Sox4 A S Sox 11 A S Sox9 cdna - GT2A G ( 2) Sox9 HM G2box 572 bp Sox 1B 6 1 ( : A Y bp 563 bp 531 bp 486 bp CS Sox9b CS Sox9a CS Sox4 http : / / ncbi1nlm1nih1gov/ BLAST/ 531 bp 486 bp Sox9

4 : Sox Sox9 683 1 Sox9 mrna DNA A. mrna B. DNA C. DNA Fig. 1 Sox9 mrna and DNA amplif ication results of Amur sturgeon and chum salmon A. Amur sturgeon mrna. B. Chum salmon DNA. C. Amur sturgeon DNA.

3 4 : Sox Sox Sox9 mrna DNA A. mrna B. DNA C. DNA Fig. 1 Sox9 mrna and DNA amplif ication results of Amur sturgeon and chum salmon A. Amur sturgeon mrna. B. Chum salmon DNA. C. Amur sturgeon DNA. 2 ( Oncorhynchus mykiss) ( Oncorhynchus keta) ( Acipenser schrenckii) Sox9 HMG2 box Fig. 2 Comparison of the deduced amino acid sequence of rainbow trout Oncorhynchus mykiss chum salmon On2 corhynchus keta and Amur sturgeon Acipenser schrenckii 98 % Sox9 100 % 85 %

4 Sox9 a Sox9 b Sox9 303 bp 258 bp http : / / www1ncbi1nlm1nih1gov/ blast/ bl2seq/ bl2. html 100 % HM G 303 bp 563 bp Sox bp http : / / www1ncbi1nlm1nih1gov/ 96 % 335 bp blast/ bl2seq/ bl21html Sox bp Sox8 98 % Sox9 384 bp Sox8 228 bp Sox4 93 % 3 Sox4 Table 1 Sequence agreement among Sox genes identif ied from 2 Sox9 HM G Amur sturgeon and chum salmon and known Sox genes from Japanese pufferf ish and zebraf ish HM G2box 100 % (Amino acid sequence agreement) Sox HM G2box DNA mrna 5 Sox The same gene 4 Genes The same gene with zebrafish with Japanese (Wagner et al ; Takamatsu et al ; pufferfish 2000 ; Zhou et al. 2002) Sox2 97 % - Sox4 98 % 98 % ( Sox4 a and Sox4 b) GT2A G Sox Sox9 98 % 98 %( Sox9 a) 100 %( Sox9 b) Sox9 Sox11 88 % 93 % HM G ( Zhou et al. Sox4 94 % 93 % ( Sox4 a and Sox4 b) 2002) Sox9 HM G Sox8 96 % Sox9 HM G 572 Sox9 a 98 % 98 %( Sox9 a) 100 %( Sox9 b) bp Sox9 Sox9 b 98 % 98 %( Sox9 a) 100 %( Sox9 b) HM G 303 bp 258 bp Sox10 97 % 98 % HM G 319 bp ( Takamat su et al. 1997) 367 bp (Zhou et al. 2002) 880 bp (Wagner et al. 1994) Sox PCR Sox9 HM G Sox Sox ( A Y A Y A Y627282) Sox9 1 Sox2 Sox4 Sox11 Sox9 ( Takif ugu rubri pes) Sox9 HM G2box vio) Sox4 a Sox4 b Sox11 Chiang et al. Sox9 (228 bp) 85 % Sox9 1 Sox HMG2box 4 Sox 5 : 97 % 98 % 88 % ( Danio re2 98 % 98 % 93 % Sox4 Sox8 (2001) Sox10 Sox4 Sox8 Sox10 a Sox9 Sox9 a Sox9 b Sox10 b 94 % 96 % 97 % Sox9 a Sox9 b 97 % Sox4 Sox4 a

5 4 : Sox Sox9 685 Sox4 b 93 % Sox10 98 % http : / / ncbi1nlm1nih1gov/ BLAST/ Sox HM G2box Sox ( References) HM G2box Bowles J Schepers G Koopman P Phylogeny of the S OX family of developmental transcription factors based on sequence and structural indicators. Dev. Biol. 227 : Chang CJ Zhou RJ Yu QX The RFL P analyses of pdsox8 and pdsox9 in two species of loaches. Hereditas (Beijing) 2000 (3) : ( In Chinese). Chang CJ Du Q Y Shao HW New progress in S OX gene fami2 ly. Hereditas (Beijing) 24 (4) : ( In Chinese). Chiang EF Pai CI Wyatt M Yan YL Postlethwait J Chung B 20011Two Sox9 genes on duplicated zebrafish chromosomes : ex2 pression of similar transcription activators in distinct sites. Dev. Bi2 ol. 231 (1) : Clarkson MJ Harley VR Sex with two S OX on : S R Y and S OX9 in testis development. Trends in Endocrinology and Metabolism 13 (3) : Foster J W Dominguez2Steglich MA Guioli S Kwok C Weller PA Stevanovic M Weissenbach J Mansour S Young ID Goodfellow PN Brook JD Schafer AJ Campomelic dysplasia and auto2 somal sex reversal caused by mutations in an SRY2related gene Sox91 Nature 372 : Galay2Burgos M Llewellyn L Mylonas CC Canario AVM Zanuy S Sweeney GE Analysis of the Sox gene family in the Euro2 pean sea bass Dicent rarchus labrax. Comp. Biochem. Physiol. Part B : Biochem. Molecular Biol. 137 (2) : Lunde K Belting HG Driever W Zebrafish pou5f1/ pou2 ho2 molog of mammalian Oct4 functions in the endoderm specification cascade. Curr. Biol. 14 (1) : Montero JA Giron B Arrechedera H Cheng YC Scotting P Chimal2 Monroy J Garcia2Porrero JA Hurle J M Expression of Sox8 Sox9 and Sox10 in the developing valves and autonomic nerves of the embryonic heart. Mechanisms of Development 118 (1-2) : Nagai K Molecular evolution of S ry and Sox gene. Gene 270 (1-2) : Pevny L H Lovell2Badge R Sox genes find their feet. Current Opinion in Genetics & Development 7 (3) : Sambrook J Fritsch EF Maniatis T Molecular cloning : a labo2 ratory manual. 2nd Edn Cold Spring Harbor Laboratory Press Chinese edition. Beijing : Science Press ( In Chinese). Schepers GE Bullejos M Hosking BM Koopman P Cloning and characterization of the S ry2related transcription factor gene Sox81 Nucleic Acids Research 28 (6) : Takamatsu N Kanda H Ito M Rainbow trout Sox9 : cdna cloning gene structure and expression. Gene 202 : Wagner T Wirth J Meyer J Autosomal sex reversal and cam2 pomelic dysplasia are caused by mutations in and around the SRY2 related gene Sox91 Cell 79 : Yue GH Orban L Rapid isolation of DNA from fresh and pre2 served fish scales for polymerase chain reaction. Mar. Biotech. 3 : Yan YL Miller CT Nissen RM Singer A Liu D Kirn A Draper B Willoughby J Morcos PA Amsterdam A Chung BC Westerfield M Haffter P Hopkins N Kimmel C Postlethwait J H Nissen R A zebrafish Sox9 gene required for cartilage morphogen2 esis. Development 129 (21) : Yokoi H Kobayashi T Tanaka M Nagahama Y Wakamatsu Y Takeda H Araki K Morohashi K Ozato K Sox9 in a teleost fish medaka Oryzias latipes : evidence for diversified func2 tion of Sox9 in gonad differentiation. Mol. Reprod. Dev. 63 (1) : Zhou RJ Cheng HH Zhang Q Y Guo YQ Cooper R K Tiersch TR SR Y2related genes in the genome of the rice field eel Monopterus albus. Genet. Set. Evol. 34 : Sox. 24 (4) : pdsox8 pdsox9 RFL P. (3) :

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