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1 动物学研究 2007, Oct. 28(5): CN /Q ISSN Zoological Research 两个蛇毒基因克隆及 cdna 序列多态性再分析 * 摘要 :α-α-bungarotoxin cdna DNA α- 5 cdna cdna α- cdna α- cdna α- cdna RNA cdna 关键词 :α- ; ; RNA ; 中图分类号 :Q959.62; Q38; Q785 文献标识码 :A 文章编号 (2007) Cloning of Two Toxin Related Genes and Analysis of Their cdna Polymorphism LIN Lu-ping, LIN Qun, WANG Yi-quan * (School of Life Sciences, Xiamen University, Xiamen , China) Abstract: Alpha-bungarotoxin is one of the post-synaptic neurotoxins, which widely exists in the Elapidae venom. The polymorphism of α-bungarotoxin s cdna sequences has been controversial in previous reports. In the present study, cloning of α-bungarotoxin gene from the genomic DNA, sequencing of five clones and analysis of their polymorphism were reported. Furthermore, we cloned and sequenced the nerve growth factor (NGF) gene from the same cdna pool as a standard, and compared the mutation rates among different sources of α-bungarotoxin cdnas, α-bungarotoxin genes and NGF cdna. The results indicated that there was no diversification in the genome or comparability in the mutative points between different sequences. Therefore, we speculate that the polymorphism of α-bungarotoxin cdna resulted neither from different transcripts, nor from RNA editing, but possibly from reverse transcript processes and gene-cloning processes. Key words: α-bungarotoxin; Polymorphism; RNA editing; Mutation α-α-bungarotoxin 1998 Liu et al 1998 cdna α- 16 mrna 10 2 α- Koesn et al,1988 V31 A31 Liu et al 1998 DNA α- cdna α- RNA Chang et al (1999) DNA 2 α D * Corresponding author wangyq@xmu.edu.cn Vanderbilt University

2 V31 A31 cdna V31 A31 α- cdna V31 A31 RNA Qian et al (2000) V31 Wang et al 2005 cdna 12 α- 8 V31 A31 α- RNA RNA RNA editing RNA Benne et al 1986 RNA Sattelle et al, 2005 RNA (Barlati et al, 2005) Hoopengardner et al (2003) 16 mrna 1 mrna RNA α- (Wu et al, 2005) α- mrna α- cdna DNA α- cdna NGF α- cdna cdna α- cdna 1 材料与方法 1.1 材料 Elapidae Bungarus multicinctus RNA RNA Wang et al, 2005 cdna RNA DNA 1.2 方法 DNA RNA DNA 20 DNA ddh 2 O 4 RNA Trizol cdna Clotech SMART TM cdna α- PCR GenBank α- DNA BgTX_genome_upper BgTX_genome_ lower α- 25 µl PCR 10 PCR Buffer 2.5 µl 10 mmol/l BgTX_genome_upper BgTX_genome_lower 0.5 µl 2.5 mmol/l dntps 4 µl 5 U/µL ExTaq 0.25 µl DNA 1 µlpcr 95 4 min s s 72 2 min 72 7 min 5 µl PCR 1.2% PCR 2 kb, TA pmd18 PCR M13-M4 M13-RV BgTX_sequencing BgTX_intronI-U BgTX_intronI-I BgTX_intron2 primer walking 1 BgTX_check_upper

3 5 : cdna 459 BgTX_check_lower α- PCR DNA α- CEQ-8000 Beckman-Coulter DNAStar- SeqMan cdna 3 RACE mrna cdna RT-PCR PCR cdna NGFu SMART (Clotech) 3 RACE CDS Ⅲ /3 PCR cdna 25 µl PCR 10 PCR Buffer 2.5 µl 10 pmmol/l NGFu CDS Ⅲ/3 PCR 0.5 µl 2.5 mmol/l dntps 4 µl 5 U/µL ExTaq 0.25 µl cdna 1 µl PCR 95 4 min s s s 72 2 min 5 µl PCR 1.2% PCR TA pmd18 PCR CEQ-8000 PolyA M13-M4 M13-RV 5 NGF_sequencing 1 DNAStar SeqMan α- = /( ) Stapleton et al (2002) 1/ / α- cdna BgTX_genome_upper BgTX_genome_lower α- 2 kb 1A cdna NGFu CDSⅢ/3 PCR cdna 900 bp 500 bp 900 bp 1B 2.2 α- α bp α- α- cdna 3 2 2A 5 α- α- 288 bp 1 58 bp N 20 1 Tab. 1 Information of theprimers used in gene cloning and sequencing Primers Sequences BgTX_genome_upper 5 -CCg gaa TTC AgA TCG CAA gat gaa AAC TC-3 BgTX_genome_lower 5 -gcg gga TCC TCA ACC Agg TCT CTg TTT C-3 BgTX_sequencing 5 -gat gaa AAA TgT TTC TgC TC-3 BgTX_intronI-U 5 -TTg gcc TTC CAT CCT TTT gaa ATg-3 BgTX_intronI-I 5 -TAg CCg AAC TAg CTg TTg TgT g-3 BgTX_intron2 5 -TTC ATC CAA TTT CCC TCA T-3 BgTX_check_upper 5 -CAA gaa gct CAC TCT gta ga -3 BgTX_check_lower 5 -TAT CAg CTC AAA AAA CCT TAA C-3 NGFu 5 -ATg TCC ATg CTg TgC TAC ACT CTg-3 CDS /3 PCR 5 -ATT CTA gag gcc gag gcg gc-3 NGF_sequencing 5 -AAg TTC CTg gac ACT gaa ga-3 M13-M4 5 -CAg CAC TgA CCC TTT Tgg gac CgC-3 M13-RV 5 -AgC gga TAA CAA TTT CAC ACA Agg-3

4 α- PCR A 3 RACE PCR B Fig. 1 Agarose gel electrophoresis of PCR amplification products of α-bungarotoxin gene (A) and 3 RACE PCR amplification products of nerve growth factor gene from the venom cdna(b) M (bp)( DL2000 marker); 1 α- (α-bungarotoxin gene); 2 cdna(nerve growth factor cdna) bp 4 N bp 41 1 V bp 538 bp 5 α- 13 2B α- cdna Wang et al A31 DNA 5 BgTX_check_upper BgTX_check_ lower DNA α- 508 bp 2A PCR PCR 5 PCR α RACE 3 RACE 6 3 RACE 891 bp 732 bp 6 11 ( 3A) 2 4 3B 6 cdna G α- PCR 5 5 cdna6 5 Wang et al(2005) α- cdna Liu et al (1998) α- Wang et al (2003) 7 cdna 2 α- cdna 2 α-a 5 α-b Fig. 2 α-bungarotoxin gene organization, distribution of mutation sites and indication of verified region A and mutation sites of five clones with α-bungarotoxin gene B

5 5 : cdna A 6 cdna B Fig. 3 Indication for the coding region and non-coding region and distribution of mutation sites of Bungarus multicinctus nerve growth factor gene A and mutation sites of six clones for nerve growth factor cdna B Gene Mutation number 2 Tab. 2 Results of mutation rates of several genes Sequenced clones Amino acid number bp Length of sequence (bp) Mutation rate Mutation rate with deducted reversal error α- α-bungarotoxin genome N/A α- cdna α-bungarotoxin cdna a cdna NGF cdna α- cdna α-bungarotoxin cdna b cardiotoxin-like gene Wang et al (2005) (a) Liu et al (1998) (b) The mutation rates calculated according to sequences from Wang et al (2005) (a) and Liu et al (1998) (b). α- cdna cdna 2.5 α- cdna α- cdna RNA α- Wang et al (2005) 12 cdna 3 9 Liu et al (1998) 16 cdna NCBI 3 cdna C1 Y17057 C2 Y17058 K X A31 3 V31 22 α- cdna T-C V31 A31 Wang et al (2005) 12 cdna Liu et al (1998) 211 C Wang et al (2005) cdna A Liu et al (1998) cdna T Wang et al (2005) cdna Liu et al (1998) cdna ( 4) 2.6 PCR α- α- cdna cdna α- cdna Liu et al, 1998 cdna 1/1000 TAKARA ExTaq 30 1 kb DNA 2 3

6 α cdna Fig.4 Alignment of exons of α bungarotoxin genomic DNA and cdna from Wang et al (W-1 W-12), Liu et al (L-R1 L-R16) and another α bungarotoxin cdna sequences from GenBank (C1, C2, K) Identical sequences are omitted. The identical nucleotides with the first line are indicated by dots and deleted nucleotides by hyphens. Wang et al(2003, 2005) rtaq 30 1 kb DNA 5 6 PCR PCR 3 α-α- cdna cdna PCR 3 讨论 3.1 α- 银环蛇毒素 cdna 多态性不是由于基因组不同转录本造成 DNA 2.65 kb α- V31 Wang et al (2003) cdna α- A31 Chang et al (1999) Wang et al (2005) α- α- V31 A31 α- 13 PCR 5 PCR α- 5 α- α- cdna α- cdna 3.2 造成 α- 银环蛇毒素 cdna 多态性的原因分析 Stapleton et al (2002) cdna Wang et al (2005) α- cdna Liu et al (1998) α- cdna Wang et al (2003) cdna cdna α- cdna cdna Liu et al (1998) α- cdna DNA α cdna cdna cdna 3 α- α- cdna

7 5 : cdna PCR Tab. 3 Results of error rates by PCR process Gene PCR Mutation points during PCR Expected mutation rate α- α-bungarotoxin genome α- cdna α-bungarotoxin cdna a cdna NGF cdna α- cdna α-bungarotoxin cdna b Cardiotoxin-like ExTaq PCR Wang et al (2005) (a) Liu et al (1998) (b) The mutation rates in sequences from Liu et al (1998) (a) and Wang et al (2005) (b) were calculated by the ExTaq-PCR error point formula. Erabutoxin Agkistrodon acutus C Endo et al,1991 Zha et al, 2004 α- Shen et al, 2000 α- Liu et al (1998) α- cdna RNA RNA (Reenan, 2005) α- cdna RNA α- cdna α- cdna RNA cdna (Yu et al,1997; Gunaratne et al, 2003) ExTaq DNA PCR PCR 1/10000 (Schmutz et al, 2004) α- cdna RNA Furey et al (2004) mrna mrna mrna RNA cdna RNA 参考文献 : Barlati S, Barbon A RNA editing: a molecular mechanism for the fine modulation of neuronal transmission [J]. Acta Neurochir Suppl, 93: Benne R, Van den Burg J, Brakenhoff JP Sloof P, Van Boom JH, Tromp MC Major transcript of the frame-shifted cox gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA [J]. Cell, 46(6): Chang L, Lin S, Huang H, Hsiao M Genetic organization of alpha-bungarotoxins from Bungarus multicinctus (Taiwan banded krait): evidence showing that the production of alpha-bungarotoxin isotoxins is not derived from edited mrnas [J]. Neucleic Acids Research, 27(20): Endo T. Tamiya N In: Harvey AL. Structure-function relationships of post-synaptic neurotoxins from snake venoms, Snake Toxins(M). New York: Pergamon Press, Furey TS, Diekhans M, Lu Y, Graves TA, Oddy L, Randall-Maher J, Hillier LW, Wilson RK, Haussler D Analysis of human mrnas with the reference genome sequence reveals potential errors, polymorphisms, and RNA editing [J]. Genome Res, 14(10B):

8 Gunaratne P, Wu J, Garcia A, Hulyk S, Worley K, Margolin J, Gibbs R Concatenation cdna sequencing for transcriptome analysis [J]. C R Biol, 326(10-11): Hoopengardner B, Bhalla T, Staber C, Reenan R Nervous system targets of RNA editing identified by comparative genomics [J]. Science 301(5634) Kosen PA, Finer-Moore J, McCarthy MP, Basus VJ Structural studies of α-bungarotoxin.3.correction in the primary sequence and X-ray structure and characterization of an isotoxin α-bungarotoxin [J]. Biochemistry, 27: Liu LF, Chang CC, Lian MY, Kuo KW Genetic characterization of the mrna encoding α-bungarotoxin : isoforms and RNA editing in Bungarus multicinctus gland cells [J]. Neucleic Acids Research, 26(24): Qian YC, Fan CY, Hu TS, Yang YG, Yang SL, Gong Y Molecular cloning and functional expression of α-bungarotoxin(v31) from Chinese continental banded krait [J].Zool Res, 21(1):41-47.[,,,,, , 21(1): ] Reenan RA Molecular determinants and guided evolution of species-specific RNA editing [J]. Nature, 434(7031): Sattelle DB, Jones AK, Sattelle BM, Matsuda K, Reenan R, Biggin PC Edit, cut and paste in the nicotinic acetylcholine receptor gene family of Drosophila melanogaster [J]. Bioessays, 27(4): Schmutz J, Wheeler J, Grimwood J, Dickson M, Yang J, Caoile C, Bajorek E, Black S, Chan YM, Denys M, Escobar J, Flowers D, Fotopulos D, Garcia C, Gomez M, Gonzales E, Haydu L, Lopez F, Ramirez L, Retterer J, Rodriguez A, Rogers S, Salazar A, Tsai M, Myers RM Quality assessment of the human genome sequence [J]. Nature, 429(6990): Shen GG, Xu K Purification of -bungarotoxin-binding protein from rat diaphragm muscle [J].Zool Res, 21(3): [ , 21(3): ] Stapleton M, Carlson J, Brokstein P, Yu C, Champe M, George R, Guarin H, Kronmiller B, Pacleb J, Park S, Wan K, Rubin GM, Celniker SE A drosophila full-length cdna resource [J]. Genome Biology, 3(12): RESEARCH Wang F Cloning and expression of the genes related to snake venom[d]. MD thesis, Nanjing NormaL University, [ , ] Wang F, Wang YQ, Tong ZZ Cloning and characterization of cdnas of cardiotoxin-like protein in Bungarus multicinctus Venom Gland[J]. Hereditas (Beijing), 25(6) [,, cdna [J]., 25(6) ] Wang F, Wang YQ Molecular cloning and expression of an isotoxin gene, α-bungarotoxin, from Bungarus multicinctus [J]. Acta Genetica Sinica, 32(7): [ α-., 32(7): ] Wu PP, Ma DD, Pierzchala M, Wu J, Yang LC, Mai XP, Chang XY, Schmidt-Glenewinkel T The drosophila acetylcholine receptor subunit Dα5 is part of an α-bungarotoxin binding acetylcholine receptor [J]. J Biol Chem, 280(22): Yu W, Andersson B, Worley K, Muzny D, Ding Y, Liu W, Ricafrente J, Wentland M, Lennon G, Gibbs R Large-scale concatenation cdna sequencing [J]. Genome Research, 7(4): Zha XD, Zhou LZ, Huang HS, Liu J, Xu KS Analysis of cdnas and genomic DNA of snake venom CTL-like proteins revealed an extraordinary post-transcriptional processing event [J]. Chinese Journal of Biochemistry and Molecular Biology, 20(6): [,,,, C cdna DNA., 20(6): ]

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