High Degree Of Sequence Identity Research Articles

Detection of aneuploidies by paralogous sequence quantification

Background: Chromosomal aneuploidies are a common cause of congenital disorders associated with cognitive impairment and multiple dysmorphic features. Pre-natal diagnosis of aneuploidies is most commonly performed by the karyotyping of...

Hypotensive Agents from Snake Venoms

Many snake venoms contain toxins which produce profound cardiovascular effects. The site of action of these toxins includes cardiac muscle, vascular smooth muscle and the capillary vascular bed. Some snake venoms, for example, contain peptides that inhibit angiotensin converting enzyme and potentiate the biological actions of bradykinin. Other snake venoms contain structural and functional equivalents of mammalian natriuretic peptides. Sarafotoxins are short peptide toxins found in the venoms of snakes from Atractaspis spp. which display potent vasoconstriction properties. These peptides, which share a high degree of sequence identity with endothelins, recognize and bind to endothelin receptors. Snakes have also evolved toxins which block L-type Ca(2+) currents (eg. calciseptine, FS2 toxins, C(10)S(2)C(2) and S(4)C(8)). Snake venom proteins have also been shown to increase vascular permeability. One such protein, increasing capillary permeability protein (ICPP) has recently been isolated from the venom of Vipera lebetina. ICPP is an extremely potent permeability factor with a structure similar to vascular endothelial growth factor (VEGF). Thus there is a vast array of snake toxins with potent cardiovascular activity. Some of these proteins and peptides have proven to be highly selective tools in the study of physiological processes. Others have been used as probes of potential therapeutic targets or as lead compounds in the development of therapeutic agents. Therefore these and other related snake venom proteins hold great promise in the future understanding and treatment of cardiovascular diseases.

In vitro and in vivo translational efficiencies of the 5′ untranslated region from eight genotype 2 bovine viral diarrhea virus field isolates

We determined the in vitro and in vivo translational efficiency mediated by the internal ribosomal entry site (IRES) from eight BVDV2 field isolates varying in virulence using a bicistronic reporter vector in rabbit reticulocyte lysates (RRL), and in primate and bovine cell lines. Using a T7-promoter system, the high virulence isolates had greater translational efficiencies in bovine lymphocytes (BL-3 cells), than did the low virulence isolates. The low virulence isolates translated with greater efficiencies than the high virulence isolates in RRL, African green monkey kidney (CV-1) and bovine turbinate (BT) cells. Our results demonstrate that despite a high degree of sequence identity in the 5′ untranslated region (UTR), subtle differences in the primary and secondary structures, as well as differences in cell lines, influence translational efficiencies.

High Expression of Micro-RNA BIC / miR155 in All Subtypes of Hodgkin Lymphoma.

We recently demonstrated that the noncoding BIC gene was highly expressed in Hodgkin Reed-Sternberg (HRS) cells of the vast majority of both classical and nodular lymphocyte predominant (NLP) Hodgkin lymphoma (HL). The BIC gene belongs to the family of noncoding mRNA-like molecules and was suggested to function through its noncoding RNA transcript. The human BIC gene shows a high degree of sequence identity to the Mouse and Gallus gallus BIC genes, which was most pronounced in a region containing a ~70 nucleotide stem-loop structure. Recently published data revealed expression of miR155 micro-RNA in murine tissue. The miR155 sequence was identical to the first 22 nucleotides of the ~70 nucleotide stem-loop structure of the BIC gene indicating that BIC is a primary micro-RNA transcript. Micro-RNAs (miRNA) are endogenous small RNAs (20–22 nucleotides) that inhibit protein translation by paring to the messages of protein-coding mRNAs. Based on recent data, it is clear that deregulation of miRNA expression, processing or stability may lead to deregulation of important cellular processes and thereby contribute to the development of cancer.In this study we analyzed the expression of miR155 in HL and NHL samples and studied the regulation of BIC/miR155 expression. Northern blot analysis revealed that HL cell lines, which all have a high level of BIC transcripts, also have high levels of the functionally mature miR155. NHL samples with low levels of BIC transcripts also have low levels of miR155. These data provide experimental support for processing of primary BIC transcripts to the functionally mature miR155 fragments and indicate that miR155 expression is specific for HL. To analyze whether BIC expression could be induced in NHL cell lines we studied several stimuli in the wild type Burkitt lymphoma derived cell line Ramos and a NF-κB deficient variant cell line. Real-time RT-PCR analysis revealed a high level of BIC and of the 70 nucleotide miR155 precursor in anti-IgM stimulated Ramos cells. Anti-IgM stimulation of the NF-κB deficient Ramos cell line revealed only low levels of BIC and pre-miR155. These data demonstrate that transactivation of NF-κB is required for continuous upregulation of BIC in Ramos cells. Stimulation of Ramos cells with PMA/calcium-ionophore, which directly activates PKC, significantly upregulated the level of BIC and pre-miR155. This suggests that activation of PKC and its downstream signaling components, including NF-κB are involved in the regulation of BIC expression. Thus, whereas HRS cells typically do not express surface immunoglobulin, upregulation of BIC in these cells may be induced by constitutively activated NF-κB.

Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium

The structural genes (hupSL) encoding an uptake hydrogenase in the unicellular cyanobacterium Gloeothece sp. ATCC 27152, a strain capable of aerobic N(2) fixation, were identified and sequenced. 3'-RACE experiments uncovered the presence of an additional ORF 184 bp downstream of hupL, showing a high degree of sequence identity with a gene encoding an uptake-hydrogenase-specific endopeptidase (hupW) in other cyanobacteria. In addition, the transcription start point was identified 238 bp upstream of the hupS translational start. RT-PCR experiments revealed that hupW is co-transcribed with the uptake hydrogenase structural genes in Gloeothece sp. ATCC 27152. In addition, Northern hybridizations clearly showed that hupSLW are transcribed under nitrogen fixing conditions, but not in the presence of combined nitrogen. A putative NtcA binding site was identified in the promoter region upstream of hupS, centred at -41.5 bp with respect to the transcription start point. Electrophoretic retardation of a labelled DNA fragment (harbouring the putative NtcA-binding motif) was significantly affected by an Escherichia coli cell-free extract containing overexpressed NtcA, suggesting that NtcA is involved in the transcriptional regulation of hupSLW.

Neprilysin 2: A Novel Messenger Peptide-Inactivating Metalloprotease

Neprilysin 2 is a recently identified glycoprotein displaying the highest degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 family of zinc-dependent metalloproteases. Whereas neprilysin has been shown to be involved in the inactivation of endogenous messenger peptides, like enkephalins and tachykinins, the true physiological functions of neprilysin 2 remain unknown.

Genetic characterisation of the Pag island sheep breed based on microsatellite and mtDNA data

Genetic variation in the Pag island sheep population was analysed using seven microsatellite loci and by sequencing of the proximal part (nt 15816–16153) of the D-loop region at mtDNA. The mean number of alleles per microsatellite locus (8.71) and mean expected heterozygosity (0.833) reveal relative high genetic variability. Sequencing of the part of the mtDNA D-loop region from 10 unrelated animals revealed 39 polymorphic sites characterizing seven haplotypes which clustered into two main haplogroups (M and P). Sequence differences among haplotype groups M and P were from 6.78 to 9.44%. Our data support the hypothesis that the M haplotype represents a trace of Merinisation of the Pag island sheep, reflected by the high degree of sequence identity with the Merino breed sequence (99%). The P haplotype belongs to the original primitive sheep that have inhabited the island of Pag for thousands of years. These findings suggest that modern Pag island sheep were derived from at least two maternal lineages, the autochthonous and imported one.

Biochemical and Molecular Characterization of a Ring Fission Dioxygenase with the Ability to Oxidize (Substituted) Salicylate(s) from Pseudaminobacter salicylatoxidans

The gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12. The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7. The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum. The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate. The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified. For comparison, the putative gentisate 1,2-dioxygenase from C. glutamicum was functionally expressed in E. coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate.

Molecular cloning, pharmacological characterization, and histochemical distribution of frog vasotocin and mesotocin receptors.

The neurohypophysial nonapeptides vasotocin (VT) and mesotocin (MT) are the amphibian counterparts of arginine vasopressin (AVP) and oxytocin (OT). We have here reported the cloning and functional characterization of the receptors for vasotocin (VTR) and mesotocin (MTR) in two species of frog, Rana catesbeiana and Rana esculenta. The frog VTR and MTR cDNAs encode proteins of 419 and 384 amino acids respectively. Frog VTR exhibits a high degree of sequence identity with the mammalian AVP-1a (V1a) receptor while the frog MTR possesses a high degree of sequence identity with the mammalian OT receptor. Activation of VTR induced both c-fos promoter- and cAMP-responsive element (CRE)-driven transcriptional activities, while activation of MTR induced c-fos promoter-driven transcriptional activity but failed to evoke CRE-driven transcriptional activity, suggesting differential G protein coupling between VTR and MTR. The VTR exhibited the highest sensitivity for VT followed by OT>AVP approximately MT, whereas the MTR showed preferential ligand sensitivity for MT>OT>VT>AVP. A V1a agonist but not V2 and OT agonists substantially activated both VTR and MTR with a similar sensitivity. V1a, V2 and OT antagonists inhibited MT-induced MTR activation but not VT-induced VTR activation. In the frog brain, VTR and MTR mRNAs were found to be widely expressed in the telencephalon, diencephalon and mesencephalon, and exhibited very similar regional distribution. In the pituitary, VTR and MTR were expressed in the distal and intermediate lobes but were virtually absent in the neural lobe. Taken together, these data indicated that, although the distribution of VTR and MTR largely overlaps in the frog brain and pituitary, VT and MT may play distinct activities owing to the ligand selectivity and different signaling pathways activated by their receptors.

Variations and taxonomic status of begomoviruses causing severe epidemics of cassava mosaic disease in Kenya, Uganda, and Democratic Republic of the Congo

The molecular variability in the DNA-A of cassava-begomoviruses, East African cassava mosaic virus (EACMV), East African cassava mosaic virus – Uganda variant (EACMV-UG), African cassava mosaic virus (ACMV), and East African cassava mosaic Zanzibar virus (EACMZV) in Kenya, Uganda, and Democratic Republic of the Congo (DRC) was investigated. Most samples from western Kenya, Uganda, and eastern DRC contained EACMV-UG. Fewer than half of the samples had a mixed infection of ACMV and EACMV-UG, and a small percentage had only ACMV infections. EACMV and EACMZV were the only begomoviruses detected in samples from the Kenyan coast. The coat protein gene nucleotide (nt) and the deduced amino acid sequence analyses revealed a high degree of sequence identity within each virus type and that EACMV-UG was most related to ACMV. However, analysis of the retrieved complete DNA-A (2781–2801 nt) sequences of selected virus types revealed that EACMV-UG DNA-A share more than 90% identity with EACMV and less than 80% with ACMV, confirming that the virus is a strain of EACMV.

Biosynthesis of the canine zona pellucida requires the integrated participation of both oocytes and granulosa cells.

In the dog, attempts to localize the expression of zona pellucida (ZP) proteins during folliculogenesis have failed to demonstrate conclusively whether any or all of the zona proteins are synthesized in the oocyte or the granulosa cells. Probing of paraformaldehyde-fixed prepubertal canine ovarian tissue sections with a panel of fluorescently conjugated lectins localized the expression of glycoproteins during folliculogenesis. We confirm that six lectins (PSA, s-WGA, ECL, GSL-II, LEL, and STL) consistently labeled the ZP and adjacent granulosa cells of the developing follicle and that canine ZP expresses beta-gal(1,4)glcNAc, beta-gal(1,3)galNac, alpha-mannose, and terminal sialic acid residues in a developmentally specific manner. Riboprobes for canine ZPA and ZPC genes were produced and used for in situ hybridization studies of mRNA expression in canine folliculogenesis. In addition, we isolated a partial cDNA transcript from total ovarian RNA for the canine ZPB gene having a high degree of sequence identity with the felid and porcine ZPB homologues. Subsequently, the ZPA gene transcripts were localized to the cytoplasm of oocytes in primordial, primary, and early secondary follicles. We then localized expression of ZPB and ZPC gene transcripts to the granulosa cells of growing follicles, but not in squamous granulosa cells of primordial follicles or oocytes. These observations indicate that in the juvenile canine ovary, the oocyte is responsible for synthesis of the ZPA protein and directing synthesis of the ZPB and ZPC proteins by the granulosa cells and that ZP gene transcription occurs in a sequential manner during folliculogenesis.

Adaptive evolution and functional divergence of pepsin gene family

In vertebrates, a large proportion of genes is organized in gene families. Paralogous gene groups generated by gene duplication are related by homology, high degree of sequence identity and similar structural architecture of their products. Aspartic proteinases form a widely distributed protein superfamily including cathepsins, pepsins, renin and napsin. In the present study, the nucleotide sequences coding for various pepsins in 30 vertebrate species have been used to derive a gene phylogeny. Gene duplication and losses have been inferred from a reconciled tree, reconstructed by combining information from gene tree and species tree. Our findings based on the results of the relative rate ratio test and maximum likelihood analysis suggest that each round of gene duplication is characterized by adaptive evolution, although instances of evolution under positive selection have been found also long after divergence of gene families. The results of functional divergence analysis provided statistical evidence for shifted evolutionary rate after gene duplication.

Cloning, expression, and structural analysis of recombinant BJcuL, a c-type lectin from the Bothrops jararacussu snake venom

The lactose-binding lectin from Bothrops jararacussu venom (BJcuL) is a homodimer belonging to group VII of the c-type animal lectins. BJcuL has also been shown to serve as an interesting tool for combating tumor progression by inhibiting cancer and endothelial cell growth. However, detailed structural studies of BJcuL and its biological mechanisms of cytotoxicity are yet to be reported, perhaps because of the non-availability of recombinant proteins in necessary quantities. Intending to increase the present information about structural and consequently the understating of biological studies, the cDNA coding for BJcuL from a venom gland has been cloned and sequenced. The mature protein-coding region was amplified by PCR with specific oligonucleotides, and subcloned into the pET-15b vector to express the recombinant BJcuL in Escherichia coli BL21 (DE3). The deduced amino acid sequence exhibits a high degree of sequence identity with c-type lectins (CTLs) and c-type lectin-like domains (CTLDs). An insoluble and inactive 18.5-kDa protein was overexpressed after 1.0 mM IPTG induction. The recombinant BJcuL was recovered and denatured in a buffer with 6 M urea and purified on a nickel-affinity column. Protein refolding was carried out on this column, during procedure purification, followed by dialysis against CTBS and then by gel filtration for separation of the active dimmer. The refolding process of rBJcuL and the analysis of its structure were confirmed by biological assay, circular dichroism, and MALDI-TOF.

Monkey corticotropin-releasing factor 1 receptor: Complementary DNA cloning and pharmacological characterization

The full-length complementary DNA (cDNA) of monkey corticotropin-releasing factor type 1 (CRF 1) receptor was isolated from a rhesus monkey ( Macaca mulatta) amygdala cDNA library. The cloned monkey CRF 1 receptor cDNA has 2,374 bp with an open reading frame encoding a 415-amino acid protein. The sequence of the monkey CRF 1 receptor cDNA showed a high degree of sequence identity with other species of CRF 1 receptors, and being 99.5% identical to human CRF 1 receptors. When monkey CRF 1 was expressed into COS-7 cells, high specific binding of [ 125I]-ovine CRF was observed. CRF and CRF-related peptides inhibited [ 125I]-ovine CRF binding in a concentration-dependent manner. IC 50 values of ovine CRF, human/rat CRF, sauvagine and urotensin I were 23.5 ± 7.4, 22.7 ± 10.8, 27.5 ± 12.3 and 14.2 ± 7.0 nM, respectively. CRF 1 receptor specific antagonists, such as CP-154,526, SC241 and CRA1000, also inhibited the [ 125I]-ovine CRF binding, with IC 50 values of 3.9 ± 0.4, 43.5 ± 8.0 and 19.8 ± 2.0 nM, respectively. GTP and its nonhydrolyzed analogue, GTPγS, reduced [ 125I]-ovine CRF binding, while ATP had a negligible effect, thereby indicating that the monkey CRF 1 receptor belongs to a family of G-protein coupled receptors. CRF and its related peptides increased cyclic AMP formation concentration-dependently in COS-7 cells transiently expressing the monkey CRF 1 receptor. Monkey CRF 1 was expressed abundantly in the pituitary, cerebral cortex, hippocampus, amygdala and cerebellum. Thus the monkey CRF 1 receptor and the human CRF 1 receptor have similar molecular and pharmacological characteristics.

Exchangeable chaperone modules contribute to specification of type I and type II Hsp40 cellular function.

Hsp40 family members regulate Hsp70s ability to bind nonnative polypeptides and thereby play an essential role in cell physiology. Type I and type II Hsp40s, such as yeast Ydj1 and Sis1, form chaperone pairs with cytosolic Hsp70 Ssa1 that fold proteins with different efficiencies and carry out specific cellular functions. The mechanism by which Ydj1 and Sis1 specify Hsp70 functions is not clear. Ydj1 and Sis1 share a high degree of sequence identity in their amino and carboxyl terminal ends, but each contains a structurally unique and centrally located protein module that is implicated in chaperone function. To test whether the chaperone modules of Ydj1 and Sis1 function in the specification of Hsp70 action, we constructed a set of chimeric Hsp40s in which the chaperone domains of Ydj1 and Sis1 were swapped to form YSY and SYS. Purified SYS and YSY exhibited protein-folding activity and substrate specificity that mimicked that of Ydj1 and Sis1, respectively. In in vivo studies, YSY exhibited a gain of function and, unlike Ydj1, could complement the lethal phenotype of sis1 Delta and facilitate maintenance of the prion [RNQ+]. Ydj1 and Sis1 contain exchangeable chaperone modules that assist in specification of Hsp70 function.

Characteristics of the Plasmodium falciparum PK5 ATP-binding site: implications for the design of novel antimalarial agents

Increasing worldwide resistance of Plasmodium falciparum ( P. falciparum) to traditional chemotherapy strategies such as chloroquine and mefloquine demonstrates the urgent need for the discovery of novel chemotherapeutic agents in the fight against malaria. The recent discovery of P. falciparum Protein Kinase 5 (P fPK5) invites the possibility of selectively targeting the life cycle of P. falciparum in order to prevent cerebral malaria. P fPK5 bears a high degree of sequence identity (>58%) to a structurally conserved family of mammalian kinases known as the cyclin-dependent kinases (CDKs). The CDKs are the key regulatory elements governing the ordered progression of the mammalian cell cycle. With numerous X-ray crystal structures of CDK2 to provide a structural template, here we present a three-dimensional structural model of P fPK5 constructed using computer-based homology modeling techniques. Our model was used to compare the ATP binding site of P fPK5 with that of the mammalian kinase CDK2. Furthermore, kinase–ligand interactions of P fPK5 with known inhibitors were investigated and compared to available crystal structures of CDK2 with inhibitors bound. The focus of the study is to identify similarities and differences between the ATP binding sites of the two kinases that can be exploited for future rational drug design.

Forssman synthetase expression results in diminished shiga toxin susceptibility: a role for glycolipids in determining host-microbe interactions.

Forssman glycolipid (FG), the product of Forssman synthetase (FS), is widely expressed among nonprimate mammalian species. Here, we describe a molecular and genetic relationship between FG expression and Shiga toxin (Stx) susceptibility. We have isolated the FS cDNA from human, canine, and murine cells. Whereas the murine and canine FS genes express a functional enzyme, the human FS cDNA was found to express a protein that lacks FS activity, despite a high degree of sequence identity with the enzymatically active murine and canine FS genes. In order to examine the relationship between FG expression and Stx susceptibility, Vero cells were transfected with the three FS orthologues or a vector control. Complementation with the human FS cDNA had no effect on Stx susceptibility, whereas stable expression of the canine and murine FS resulted in markedly decreased susceptibility to toxin. Among individual cells, an inverse correlation between FG expression and Stx binding was demonstrated. Moreover, only strongly FG-reactive cells were capable of growing in the presence of Stx. These cells were found to have high levels of FG expression and a correspondingly diminished GbO(3) content. We conclude that expression of a functionally active FS modifies Stx receptor glycolipids to FG and results in markedly decreased susceptibility to toxin. We speculate that inactivation of the FS gene during primate evolution may account, at least in part, for the marked susceptibility of human cells to Stx.

Solution structure of Pi4, a short four-disulfide-bridged scorpion toxin specific of potassium channels.

Pi4 is a short toxin found at very low abundance in the venom of Pandinus imperator scorpions. It is a potent blocker of K(+) channels. Like the other members of the alpha-KTX6 subfamily to which it belongs, it is cross-linked by four disulfide bonds. The synthetic analog (sPi4) and the natural toxin (nPi4) have been obtained by solid-phase synthesis or from scorpion venom, respectively. Analysis of two-dimensional (1)H NMR spectra of nPi4 and sPi4 indicates that both peptides have the same structure. Moreover, electrophysiological recordings of the blocking of Shaker B K(+) channels by sPi4 (K(D) = 8.5 nM) indicate that sPi4 has the same blocking activity of nPi4 (K(D) = 8.0 nM), previously described. The disulfide bonds have been independently determined by NMR and structure calculations, and by Edman-degradation/mass-spectrometry identification of peptides obtained by proteolysis of nPi4. Both approaches indicate that the pairing of the half-cystines is (6)C-(27)C, (12)C-(32)C, (16)C-(34)C, and (22)C-(37)C. The structure of the toxin has been determined by using 705 constraints derived from NMR data on sPi4. The structure, which is well defined, shows the characteristic alpha/beta scaffold of scorpion toxins. It is compared to the structure of the other alpha-KTX6 subfamily members and, in particular, to the structure of maurotoxin, which shows a different pattern of disulfide bridges despite its high degree of sequence identity (76%) with Pi4. The structure of Pi4 and the high amounts of synthetic peptide available, will enable the detailed analysis of the interaction of Pi4 with K(+) channels.

Molecular characterization of Trimeresurus stejnegeri venom l-amino acid oxidase with potential anti-HIV activity

A novel l-amino acid oxidase, named TSV-LAO, has been purified and cloned from the snake Trimeresurus stejnegeri. Fifty percentage cytotoxic concentrations (CC 50) of TSV-LAO on C8166 cells were 24 and 390 nM in the absence or presence of catalase (400 nM), respectively. However, at concentrations that showed little effect on cell viability, TSV-LAO displayed dose dependent inhibition on HIV-1 infection and replication. The antiviral selectivity indexes (CC 50/EC 50) were 16 and 6, respectively, corresponding to the measurements of syncytium formation and HIV-1 p24 antigen expression. Interestingly, the presence of catalase resulted in an increase of its antiviral selectivity to 52 and 38. Under the same conditions, no anti-HIV-1 activity was observed by exogenous addition of H 2O 2. The complete amino acid sequence of TSV-LAO, as deduced from its cDNA, exhibits a high degree of sequence identity with other snake venom LAOs.

Novel neurotoxins from Taiwan banded krait ( Bungarus multicinctus) venom: purification, characterization and gene organization

Two novel neurotoxins BM10-1 and BM10-2 were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combinations of chromatography on a SP-Sephadex C-25 column and a reverse phase HPLC column. BM10-1 contained 66 amino acid residues including 10 Cys residues, while BM10-2 consisted of 65 amino acid residues with 8 Cys residues. The secondary structure of both BM10-1 and BM10-2 was dominated with β-sheet, but their gross conformation differed as evidenced by CD spectra and acrylamide quenching studies. BM10-1 inhibited carbachol-induced muscle contraction in a reversible manner and the dose for achieving 50% inhibition was approximately fourfold that of α-bungarotoxin. BM10-2 exhibited an irreversible but weak inhibition on carbachol-induced muscle contraction. Sequence alignment of neurotoxins with BM10-1 and BM10-2 suggested that the manner in the manifestation of their activity could be partly elucidated by the residues at toxin second loop. The genomic DNAs encoding BM10-1 and BM10-1-like protein (BM10-1L) were amplified by PCR. The two genes shared virtually identical structural organization and high degree of sequence identity with B. multicinctus neurotoxin genes. Compared to intron sequences of these genes, the protein-coding regions were highly variable. The difference between BM10-1 gene and BM10-1L gene notably arose from the third exon. These results suggest the evolution of B. multicinctus neurotoxins via the path of gene duplication.