Molecular cloning of a cardiotoxin structural gene from Malayan spitting cobra (Naja naja sputatrix)

Yeo, Mei-Shi; Jeyaseelan, Kandiah; Chung, Maxey C. M.; Gopalakrishnakone, P.; Tan, C.H.; Wong, H.A.

doi:10.1016/0041-0101(93)90356-n

Cited by 14 publications

(6 citation statements)

References 13 publications

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“…Total RNA was isolated from the venom glands using the guanidine isothiocyanate method [19] and the integrity of the total RNA was analyzed by denaturing formaldehyde agarose electrophoresis [20]. Oligonucleotide primers synthesized by Oswel DNA service (UK) based on the sequence at the 5′ and 3′ UTR regions of the previously cloned cDNAs [16,21] from a spitting cobra, Naja naja sputatrix have been used. The sense and antisense primers were: X133 (5′‐TCCAgAAAAagATCgCAAgATg‐3′) and X132 (5′‐gAATTTAgACATTATCAgTTg‐3′), respectively.…”

Section: Methodsmentioning

confidence: 99%

Postsynaptic short‐chain neurotoxins from Pseudonaja textilis

Gong

Armugam

Jeyaseelan

1999

European Journal of Biochemistry

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Two lethal proteins, which specifically bind to the nAChR from Torpedo californica, were isolated from the venom of Pseudonaja textilis, the common brown snake from Australia. The isolated proteins have masses of 6236 and 6345 Da and are structurally related to short-chain neurotoxins from other elapids. Six cDNAs encoding isoforms of related neurotoxins were cloned using the RT-PCR of the venom gland mRNAs. The sequences of the corresponding proteins consist of 57±58 amino acid residues and display several unique features when compared with all known short-chain neurotoxins. Accordingly, they grouped separately in phylogenetic analysis. The six cDNAs were expressed in Escherichia coli and the recombinant proteins were characterized. They have similar masses and display similar toxicities and binding constants to the nAChR as the native toxins isolated from the venom. Thus, a new group of short-chain postsynaptic neurotoxins from the venom of an Australian elapid has been characterized.

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Section: Methodsmentioning

confidence: 99%

Postsynaptic short‐chain neurotoxins from Pseudonaja textilis

Gong

Armugam

Jeyaseelan

1999

European Journal of Biochemistry

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“…These cardiotoxins are highly conserved proteins and exhibit more than 90% amino acid sequence homology [6]. The very first report on cloning of a cardiotoxin cDNA was published by our laboratory [7]and since then about a dozen cardiotoxins and cardiotoxin‐like cDNA sequences have been reported [8, 9]and/or deposited in the GenBank database by other laboratories.…”

Section: Introductionmentioning

confidence: 99%

Structure and organization of the cardiotoxin genes in Naja naja sputatrix¹

et al. 1998

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We report the genomic structure, organization and the presence of multiple isoforms of the gene encoding cardiotoxins (CTX) of Naja naja sputatrix. The cardiotoxin gene consists of six CTX isoforms, each (2.2 kb) having three exons and two introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP-2, NFIL-6/C/EBP, NF-kappaB and PuF have been identified in the 5'-region of the gene. The CTX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3, which correspond to the functional domains in the three-finger loop structure of the cardiotoxin molecule. The diverse functions of cardiotoxins together with our findings suggest that the cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process.

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“…Oligonucleotide primers synthesized by the Oswel DNA Service (Southampton, U.K.) based on sequence at the 5h and 3h untranslated regions (UTRs) of the previously cloned cDNAs from a spitting cobra, Naja naja sputatrix [22,23], were used. The sense and antisense primers were : X133 (5h-TCCAGAAAAAGATCGCAAGATG-3h) and X132 (5h-GAATTTAGACATTATCAGTTG-3h), respectively.…”

Section: Isolation Of Total Rna and Reverse-transcriptase Pcr (Rt-pcr)mentioning

confidence: 99%

Cloning and characterization of the pseudonajatoxin b precursor

GONG

Armugam

Mirtschin³

et al. 2001

Biochemical Journal

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An Australian common brown snake, Pseudonaja textilis, is known to contain highly lethal neurotoxins. Among them, a long-chain α-neurotoxin, pseudonajatoxin b, has been identified. In this report, while presenting evidence for the presence of at least four such long-chain α-neurotoxins in the venom of P. textilis, we describe the characteristics of both the mRNA and the gene responsible for the synthesis of these neurotoxins. A precursor toxin synthesized from the gene has been identified as being capable of producing the isoforms possibly by post-translational modifications at its C-terminal end. Recombinant toxins corresponding to the precursor and its product have been found to possess similar binding affinities for muscular acetylcholine receptors (IC50 = 3×10−8 M) and a lethality, LD50, of 0.15μg/g in mice.

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Molecular cloning of a cardiotoxin structural gene from Malayan spitting cobra (Naja naja sputatrix)

Cited by 14 publications

References 13 publications

Postsynaptic short‐chain neurotoxins from Pseudonaja textilis

Postsynaptic short‐chain neurotoxins from Pseudonaja textilis

Structure and organization of the cardiotoxin genes in Naja naja sputatrix¹

Cloning and characterization of the pseudonajatoxin b precursor

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Molecular cloning of a cardiotoxin structural gene from Malayan spitting cobra (Naja naja sputatrix)

Cited by 14 publications

References 13 publications

Postsynaptic short‐chain neurotoxins from Pseudonaja textilis

Postsynaptic short‐chain neurotoxins from Pseudonaja textilis

Structure and organization of the cardiotoxin genes in Naja naja sputatrix1

Cloning and characterization of the pseudonajatoxin b precursor

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Structure and organization of the cardiotoxin genes in Naja naja sputatrix¹