Cloning and sequencing of the AaH I′ gene

Cloning and sequencing of the AaH I′ gene

F.kvenW European Mating 393 characterization of molecules such as nAchRa and VOCCa, which have fundamental roles is the control of hormone release f...

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F.kvenW European Mating


characterization of molecules such as nAchRa and VOCCa, which have fundamental roles is the control of hormone release from SCLC cells and, possibly, cell proliferation as well . Pw/f+catiorr acrd characterization of two trorxl peptide toxins froth the varotrr of the acorplott Bathos tamulus which nrhibit putatkx voltage-acrfaatedpotassGen channsLr . F. A. Ds-Ar.[.~ and P. N. Sraot~ta (Neuromuscular Unit, Royal Postgraduate Medical School, Hammersmith Hospital, London Rt12 ONN, U.K .) . Two xov~ . peptide toxins have been purified to homogen«ty from the venom of the scorpion Bathos tanrrthts by a combination of ion~xchaage and reverse phase chromatography . They have been characterised using toxin binding assays to membranes from bovine brain and functional ~Rb+ influx assays in a rat glioma cell line (De-Arr m and Srnoxo, aubmittod) . Toxin 1 is a single chain peptide (mol. wt 399 and shows sequence homology to other scorpion derived inhibitors of Cap+-activated K+ channels (63% keliotoxin, 40% charybdotoxin). Toxin 2 is considerably larger; although it is only partially sequenced, there appears to be wnsiderable homology with a neurotoxin from Aplysia. Both peptides fully inhibit '~I~harybdotoxin and 'pI~eadrotoxin binding to bovine brain membranes wiW high affinity, although they have no effect oa 'uI-apamin binding to guinea-pig liver membranes Scatchard analysis indicates that the two peptides act as non-competitive inhibitors of both dendrotoxin and charybdotoxin. Unlike charybdotoxin, which speciscally inhibits ionomycdn-activated ~Rb+ influx into C6 rat glioma cells (rc, s = 0.3 n11~, neither of the new peptides has any inhibitory affect on °~Rb+ influx . (Ionomycin-stimulated ~Rb+ influx is also insensitive to dendrotoxin and apamin.) Our data sug~st that these two peptides act in a similar manner to deadrotoxin and probably inhibit voltago-activated potassium channels . The peptides interact at sites distinct from charybdotoxia and dendrotoxin in bovine brain membranes and modulate the binding of these latter two toxins by allosteric mechanisms. Pieptide 1 is distinctive in that although it is highly homologous to known Ca'+-activated K+ charm« toxins, its physiological properties resemble those of voltage-activated potassium charm« toxins . It seems lik«y that these two novel peptides define at least one new class of peptidyl inhibitor of voltago-activated potassium channels. CJonürg and sequatcing of the dafl l' gare. M. L. r~ ".~ and P. E. Bouars (CNRS, URA 1435, Université d'Aix- Marseille II, Laboratoire de Biochimie, Faculté de Médxine Secteur Nord, Hd. Pierre Dramard, 13326, Marseille, France). A t.maeteY (titre: 1.3 x lOspfu/ml) of .lndroctonus australLs geaorpic DNA was constructed in bacteriophage 1 FIX (atratageae). 9 x 10' recombinant clones were plated and transferred onto nylon filters and hybridised with radiolabelled AaH I precureor cDNA. One positive clone wan isolated and the gene structure was amplified by PCR technica. The primers corresponded to Banking sequeacea of precursor AaH I cDNA. A single band of around 800 by was obtained, subcloned into M18mp18/19 and sequenced by the Sanger's method . The sequence revealed that the AaH I' gene was cloned (AaH I' differs from AaH I by the Valise residue 17 of AaH I replaced by an Isoleuciae in AaH I~. AaH I' gene contains two axons (axon 1 = 48 by and axon 2 = 207bp) and one intros = 426by near the end of the signal peptide sequence of the toxin precursor. Nucleotide sequence and structure analysis ojthe lethal nturotoxbt Tx 1 from the unom glmtd oftlu BraziJJart spider Phoneutria nigriveater. M. R. V. Dn~trz,'~ M. J. I. Peaas,~ C. R. Du~uz,' R. D. G. Tr~+xsrox= and J. M. Cru~mx' ('Fundaçâo Ezequiel Dial, BR; =Venom Research Unit, and'Molecular Genetics Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA, U.K .). Tt~ v~woM of the Brazilian spider Phorreutrla nigriventer is mainly aeurotoxic, containing several neurotoxic peptides . A cDNA library was constructed from the venom glands and a clone coding for Txl, a lethal toxin, was identified and sequenced. The sequence data derived from this cDNA clone combined with the previously determined amino acid sequence predict that Txl ie initially ayutheaised as a preproprotein. Four segments, comprising the signal sequence, a short glutamate-rich sequence with 13 amino acids, the functional toxin and two glycine residues can be distinguished . The structure of the preprotoxin and the proposed Processing steps requirsd to form the mature Txl toxin show similarities with the synthesis and processing of m-agatoxin from funs« web-spider (Agelettopsis spans) venom. This research was funded by CNPq (Hratilian Research Council), the Medical Research Council and the Welkwme Trust, U.K. JMC is a Wellcome Trust Senior Research Fellow is Basic Biomedical Sciences . New moJfusc-specifu a-covtotoxins target neurons! acetylcholine nceptors. M. F~~u~rr~r-~ ,' A. H~ox,= R. OxEtv,~ A. L. Buxr .nla~,' D. Goanox,' M. E. Srau 2 and E. Za .mxna' (Departments of 'Cell & Animal Biology, and 2Neurobiology, Silberuraa Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel ; and'Mesa Spectrometry Facility, School of Pharmacology, University of California, San Francisco, CA, U.S .A.) . Two t~xoroxrc peptides have been isolated and purified from the venom of the molluscivorous snail Carats pennaceus. These new toxins cause strong muscle contractions and looomotory paralysis upon injection to limpet (PateJJa) snails, but have ao toxic effects in vertebrate or insect bioassays. The two new peptides are isotoxias designated coaotoxins PnIA and PnIB. They are single chain Gtetminal amidated 16 amino acid peptides, with four cyst«ne residues arranged in the characteristic framework of a-conotoxins . The amino acid sequences of ~rox u~-s