RESUMO
We examined the influence on the interferon (IFN) signaling pathway of infection with herpes simplex virus type 1 (HSV-1) strain VR3. Data from reporter gene assays showed that expression of both type I and type II IFN-inducible genes was dramatically suppressed during the early stage of HSV-1 infection (2 to 3 h postinfection). During these periods, phosphorylation levels of janus kinases (JAKs) and STATs did not increase after treatment of HSV-1-infected FL cells with IFN-alpha or IFN-gamma, although cellular protein levels of the JAKs and the STATs were not significantly changed. In contrast, the inhibitory effect of HSV-1 on phosphorylation of STAT1 was not observed in U937 cells, which show resistance to steady-state accumulation of RNA for HSV-1 immediate-early genes. The phosphorylation of STAT1 in FL cells was not inhibited by infection with a UV-inactivated virus. These results indicate that viral gene expression or viral protein production is necessary for the inhibition of phosphorylation by HSV-1.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Interferons/fisiologia , Proteínas Proto-Oncogênicas , Transativadores/fisiologia , Herpes Simples/metabolismo , Humanos , Janus Quinase 1 , Janus Quinase 2 , Fosforilação , Proteínas Tirosina Quinases/fisiologia , Fator de Transcrição STAT1 , Transdução de Sinais , Células U937 , Replicação ViralRESUMO
It has been reported that interferon (IFN)-alpha/gamma signal transduction pathway is blocked in several cell lines persistently infected with mumps virus (MV) through decrease of STAT-1alpha. Expression of the MV structural V protein (MV-V) or C terminal CYS-RICH region of the V protein (MV-Vsp) inhibited the establishment of the antivirus state induced by IFN, but not by expression of the MV-P protein. Suppression of IFN-induced STAT-1alpha, STAT-2, and IRF-9 (p48) induction was also recognized in the cells transfected with expression vector of the MV-V (pTM-V) or MV-Vsp (pTM-Vsp) protein, even though it was in the absence of the other virus protein. It is supposed that the cysteine-rich domain of V protein (Vsp) is involved in the suppression of the IFN signal transduction pathway.
Assuntos
Interferon-alfa/antagonistas & inibidores , Interferon gama/antagonistas & inibidores , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Western Blotting , Linhagem Celular , Chlorocebus aethiops , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Células Clonais/virologia , Proteínas de Ligação a DNA/metabolismo , Fator Gênico 3 Estimulado por Interferon , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Interferon gama/metabolismo , Interferon gama/farmacologia , Vírus da Caxumba/fisiologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Fator de Transcrição STAT2 , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Transfecção , Células Vero , Ensaio de Placa Viral , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/farmacologiaRESUMO
Cells of the human promonocytic cell line U937 were found to be sensitive to hexadecylphosphocholine (HPC), which is a potential anticancer drug. Induction of apoptosis was found in U937 cells after treatment with HPC for 24 to 48 hr. The apoptosis in U937 cells exposed to HPC was increased significantly in the presence of interferon-gamma (IFN-gamma). The augmentation of HPC-induced apoptosis by IFN-gamma is repressed in cells (U937-MP) persistently infected with mumps virus. A persistently infected cell line, U937-MP, showed poor induction of signal transducers and activators of transcription-1alpha (STAT-alpha), STAT-2, p48 and IFN-regulatory factor-1 (IRF-1), which are closely correlated with interferon-alpha (IFN-alpha) and IFN-gamma signaling pathways. Expression of MHC class-I or class-II was augmented by IFN-alpha or IFN-gamma in U937 cells, but not in persistently infected cells. Therefore, it is suggested that the IFN-gamma signaling pathway plays an important role in the augmentation of HPC-induced apoptosis. Mumps virus can suppress the IFN-gamma signaling pathway and subsequent development of apoptosis.
Assuntos
Apoptose , Interferon gama/metabolismo , Vírus da Caxumba/metabolismo , Fosforilcolina/análogos & derivados , Transdução de Sinais , Proteínas de Ligação a DNA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Fator Regulador 1 de Interferon , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Interferon gama/farmacologia , Vírus da Caxumba/fisiologia , Fosfoproteínas/metabolismo , Fosforilcolina/metabolismo , Fosforilcolina/farmacologia , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Células U937RESUMO
Persistent infections with mumps virus were established in human B-lymphoid cell line Akata and in the human chronic myelogenous leukaemia cell line K562. Even after IFN treatment a drastic decrease in STAT-1alpha (signal transducers and activators of transcription-1alpha), STAT-2 and p48 (ISGF-3gamma: IFN-stimulated gene factor-3gamma), which are closely correlated with the IFN-signaling pathway, was found in these persistently infected cells (Akata-MP1 and K-MTP). Therefore, the IFN-signaling pathway is thought to be defective in these persistently infected cells. In other words, most of the IFN-inducible genes in these cells persistently infected with mumps virus may not be able to respond to IFN treatment. Indeed, poor induction of 2',5'-oligoadenylate synthetase (2-5AS), dsRNA activated protein kinase (PKR), and MxA protein mRNAs were demonstrated in these cell lines after IFN treatment. Expression of MHC class-I antigen was also significantly reduced in the persistently infected cell lines as compared with that of uninfected control cells. HLA antigen was augmented by IFN-alpha in Akata and K562 cells, but not in persistently infected cells. Furthermore, suppression of IFN-induced 2-5AS induction and MHC class-I expression was restored by treatment of persistently infected cells with ribavirin through inhibition of virus replication. The result of restoration was also confirmed by IFN-induced STAT-1 induction in persistently infected cells treated with ribavirin.
Assuntos
Regulação da Expressão Gênica , Interferons/fisiologia , Vírus da Caxumba/efeitos dos fármacos , Vírus da Caxumba/metabolismo , Ribavirina/farmacologia , Fatores de Transcrição/biossíntese , Antivirais/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/virologia , Proteínas de Ligação a DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator Gênico 3 Estimulado por Interferon , Células K562 , Vírus da Caxumba/crescimento & desenvolvimento , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Verotoxin type 2 (VT2) produced by enterohemorrhagic Escherichia coli (EHEC) has been shown to have high cytotoxic potency toward several human B lymphoid cell lines with and without Epstein-Barr virus (EBV). Cell death, apoptosis induced by VT2, is closely correlated with the expression of receptor molecule Gb3/CD77, recognized by the toxin, but not with the infection or presence of EBV. Pretreatment of cells with interferon-alpha (IFN-alpha) for 24 h resulted in augmentation of apoptosis by VT2. Pretreatment within 8 h, however, was not effective. It has been reported that IFN-alpha-induced apoptosis is correlated with the induction of the 2',5'-OAS/RNase L system or dsRNA-activated protein kinase (PKR) or both. We have established persistent infection in both Akata and P3HR-1 cells with mumps virus. The persistently infected cell lines, P3HR-MP2 and Akata-MP2, showed poor induction of 2',5'-OAS and PKR in response to IFN-alpha. Augmentation of VT2-induced apoptosis by IFN-alpha was not found in the cell lines P3HR-MP2 and Akata-MP2. Therefore, these findings were interpreted to indicate that augmentation of VT2-induced apoptosis by IFN-alpha may be mediated by PKR and the 2',5'-OAS/RNaseL system. It is also suggested that mumps virus can suppress apoptosis and establish persistent infection.
Assuntos
Antivirais/uso terapêutico , Toxinas Bacterianas/toxicidade , Citotoxinas/toxicidade , Escherichia coli , Interferon-alfa/uso terapêutico , Vírus da Caxumba , Caxumba/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Doença Crônica , Sinergismo Farmacológico , Genoma Viral , Herpesvirus Humano 4/genética , Humanos , Toxina Shiga II , Transdução de Sinais/efeitos dos fármacosAssuntos
Infecções por Vírus de DNA/imunologia , Vírus de DNA/fisiologia , Interferons/fisiologia , Infecções por Vírus de RNA/imunologia , Vírus de RNA/fisiologia , Apoptose , Proteínas de Ligação a DNA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Janus Quinase 1 , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Poor induction of interferon-induced 2',5'-oligoadenylate synthetase (2-5AS) has been demonstrated in cells persistently infected with mumps virus as compared with uninfected cells. As for the number of interferon (IFN) receptors and the level of IFN regulatory factors (IRF-1 and IRF-2) mRNAs, there was little difference between them. Therefore, it is suggested that the IFN-alpha signaling system is ineffective in the persistently infected cells. Components of IFN-stimulating gene factor 3 alpha (ISGF-3 alpha), STAT-1 alpha (p91) and STAT-2 (p113), were investigated in human amnion (FL), human nasopharyngeal cancer (KB), human T-lymphoid (HUT 78), and human B-lymphoid (Akata) cells persistently infected with mumps virus. STAT-1 alpha, but not STAT-2, disappeared in these persistently infected cells, and this factor was not restored by treatment of these cells with IFN. However, no difference was observed between the levels of STAT-1 alpha mRNA transcript in persistently infected and uninfected control cells. It is reasonable to infer that the poor induction of 2-5AS activity is due to the decrease of STAT-1 alpha in correlation with the IFN-signal transduction pathway. Furthermore, induction of other IFN-stimulated genes (ds-RNA activated protein kinase, PKR, and MxA protein) was also reduced in the cells persistently infected with mumps virus.
Assuntos
2',5'-Oligoadenilato Sintetase/biossíntese , Proteínas de Ligação a DNA/fisiologia , Interferons/farmacologia , Vírus da Caxumba/fisiologia , Transativadores/fisiologia , Linhagem Celular , Indução Enzimática , Humanos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1 , Fator de Transcrição STAT2RESUMO
We demonstrated that botulinum neurotoxin attenuated the spontaneous beating rate of cultured cardiac myocytes. Primary cultured cardiac myocytes were prepared from the ventricles of neonatal Wistar rats (1-3 days old). On 7 days after cell seeding, botulinum toxin type A incorporated into liposomes was added to the culture medium. At a final concentration of 5.0 micrograms/ml, botulinum toxin markedly attenuated the beating rate of cardiac myocytes within 2-4 hours. These results demonstrated the effect of SNARE-complex proteins on the spontaneous beating of cardiac myocytes.
Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Contração Miocárdica/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Toxinas Botulínicas Tipo A/imunologia , Resinas de Troca de Cátion/farmacologia , Células Cultivadas , Depressão Química , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/imunologia , Lipídeos/farmacologia , Contração Miocárdica/imunologia , Miocárdio/citologia , Ratos , Ratos WistarRESUMO
Seventeen monoclonal antibodies (MAbs) were previously established against the heavy chain (Hc) of botulinum type E neurotoxin in BALB/c mice immunized with the type E toxoid. Five MAbs (LE15-5, LE34-6, EK19-7, EK21-4, and AE27-9) showed toxin-neutralizing activity in mice. Two of the five MAbs, EK19-7 and EK21-4, recognized the regions located at amino acid positions 731 to 787 and 811 to 897, respectively. One of the remaining three antibodies (LE34-6) reacted with the amino acid sequence VIKAIN, at amino acid positions 663 to 668, closed by the ion channel-forming domain. It is suggested that the ion channel-forming domain may also be associated with the blocking of acetylcholine release. Furthermore, the amino acid sequence YLTHMRD within 30 residues of the C-terminal region of the Hc component seemed to be recognized by LE15-5. It has been reported that the binding domain of the type E toxin is located on the C-terminal half of the Hc component. Therefore, the neutralizing activity of LE15-5 antibody may be attributed to its ability to block the binding of neurotoxin to the receptor of target cells.
Assuntos
Toxinas Botulínicas/imunologia , Clostridium botulinum/imunologia , Epitopos/genética , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Toxinas Botulínicas/genética , Clostridium botulinum/genética , Mapeamento de Epitopos , Epitopos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
Some viruses seem to be capable of suppressing interferon (IFN)-induced 2',5'-oligoadenylate synthetase (2-5AS) induction. Cells infected with human T-lymphotropic virus type-I (HTLV-I) show different natures for the constitutive production of IFN-gamma or sensitivity to IFN. Poor induction of 2-5AS was found in IFN-gamma producer cells carrying HTLV-I (MT-1, MT-2 and SMT-1). On the other hand, in non- or low-producing cell lines of IFN-gamma such as HUT102 and OKM-2, significant activity of 2-5AS was induced by treatment with IFN-alpha. A satisfactory level of IFN receptor was detectable in SMT-1 cells in spite of the poor induction of 2-5AS. There were no differences in either the interferon regulatory factor-2 (IRF-2) mRNA transcript or the level of STAT-1 alpha between SMT-1 and HUT102 cells. However, the transcription of IRF-1 mRNA was slightly reduced in SMT-1 cells as compared with that of HUT102 cells.
Assuntos
2',5'-Oligoadenilato Sintetase/genética , Proteínas de Ligação a DNA/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Fosfoproteínas/genética , Receptores de Interferon/genética , Proteínas Repressoras , Transativadores/genética , Fatores de Transcrição , Northern Blotting , Indução Enzimática , Regulação Enzimológica da Expressão Gênica , Humanos , Fator Regulador 1 de Interferon , Fator Regulador 2 de Interferon , Proteínas de Membrana , RNA Mensageiro , Receptor de Interferon alfa e beta , Fator de Transcrição STAT1 , Células Tumorais CultivadasRESUMO
Melanophores of the isolated tail fin of the Xenopus tadpole aggregate melanin granules in response to light. This aggregation was found to be inhibited by subcutaneous injection of exoenzyme C3 of Clostridium botulinum. A 26 kDa protein in homogenate obtained from the Xenopus tail fin was ADP-ribosylated by exoenzyme C3. This reaction was inhibited effectively by a monoclonal antibody, anti-Rho mab A5. raised against the small GTP-binding protein Rho. The extent of ADP-ribosylation depended on light and guanine nucleotide. Incubation under illumination partly reduced ADP-ribosylation and the reduction was restored by addition of guanine nucleotide during incubation. These findings suggest that Rho is involved in the photo-sensitive melanophore response as a signal transducer linking photo-stimuli to melanin granule translocation with Xenopus melanophores.
Assuntos
Toxinas Botulínicas , Luz , Melaninas/metabolismo , Melanóforos/fisiologia , ADP Ribose Transferases/farmacologia , Adenosina Difosfato Ribose/metabolismo , Animais , Proteínas de Ligação ao GTP/imunologia , Melanóforos/efeitos dos fármacos , Cauda/química , Cauda/citologia , Cauda/efeitos dos fármacos , Xenopus laevis , Proteína rhoA de Ligação ao GTPRESUMO
Poor induction of interferon-induced 2', 5'-oligoadenylate synthetase (2-5AS) activity has been demonstrated in cells persistently infected with the mumps virus or human T-lymphotropic virus type-I (HTLV-I). The suppression of 2-5AS induction is the result of the repression of 2-5AS gene expression at the transcription level. In a general way, after the binding of interferon-alpha (IFN-alpha) to cell surface-specific receptors, expression of 2-5AS gene is thought to be regulated by some transacting factors, IFN-regulatory factors (IRF-1 and IRF-2) and the IFN-stimulated gene factor (ISGF-3, a complex consisting of STAT-1 alpha, STAT-2 and p48). To clarify the cause of the suppression mechanism(s), fluctuation in the number of IFN receptors and the levels of mRNAs in both IRF-1 and IRF-2 were examined in cells persistently infected with the mumps virus (FLMT and KBMT). There were few differences in the number of IFN receptors and the level of IRF-2 mRNA between persistently infected cells and uninfected control cells. After the treatment of cells with IFN, a slight reduction of IRF-1 mRNA was found in persistently infected cells as compared with that of the uninfected control cells.
Assuntos
2',5'-Oligoadenilato Sintetase/biossíntese , Proteínas de Ligação a DNA/biossíntese , Vírus da Caxumba/fisiologia , Fosfoproteínas/biossíntese , Receptores de Interferon/metabolismo , Proteínas Repressoras , Fatores de Transcrição/biossíntese , 2',5'-Oligoadenilato Sintetase/genética , Linhagem Celular , Indução Enzimática , Humanos , Fator Regulador 1 de Interferon , Fator Regulador 2 de Interferon , Interferon Tipo I/metabolismo , Interferon Tipo I/farmacologia , Proteínas de Membrana , Reação em Cadeia da Polimerase , Receptor de Interferon alfa e beta , Proteínas Recombinantes , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Botulinum neurotoxin C1 inhibited Ca(2+)-evoked norepinephrine secretion from digitonin-permeabilized PC12 cells. The inhibition by the neurotoxin was dependent on the presence of Zn2+ added exogenously. This zinc-dependent inhibition was neutralized by monoclonal antibodies that recognize the sites close to the putative zinc-binding motif in the light chain. The neurotoxin was found to have an endopeptidase activity toward small peptide, substance P. The presence of exogenous Zn2+ was also indispensable to the full expression of this endopeptidase activity. Thus both the inhibition of neurotransmitter release by the C1 neurotoxin and its endopeptidase activity are dependent on exogenous Zn2+, which suggests a strong link between the two activities.
Assuntos
Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/farmacologia , Cálcio/farmacologia , Endopeptidases/metabolismo , Neurotoxinas/farmacologia , Norepinefrina/metabolismo , Substância P/metabolismo , Zinco/farmacologia , Neoplasias das Glândulas Suprarrenais , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Toxinas Botulínicas/antagonistas & inibidores , Cinética , Dados de Sequência Molecular , Neurotoxinas/metabolismo , Células PC12 , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Feocromocitoma , Ratos , Substância P/química , Especificidade por SubstratoRESUMO
The complete nucleotide and deduced amino acid sequence of the nontoxic component of botulinum type E progenitor toxin is determined in recombinant plasmid pU9BUH containing about 6.0 kb HindIII fragment obtained from chromosomal DNA of Clostridium butyricum strain BL6340. The open reading frame (ORF) of this nontoxic component gene is composed of 3,486 nucleotide bases (1,162 amino acid residues). The molecular weight calculated from deduced amino acid residues is estimated 13,6810.1. The present study revealed that 33 nucleotide bases of 3,486 are different in the nontoxic component gene between C. butyricum strain BL6340 and C. botulinum type E strain Mashike. This corresponds to the difference of 17 amino acid residues in these nontoxic component.
Assuntos
Toxinas Botulínicas/genética , Clostridium botulinum/genética , Clostridium/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Clostridium/classificação , DNA Bacteriano/genética , Expressão Gênica , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Plasmídeos/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
We have analysed the genes borne on a 6.0 kb HindIII fragment cloned from the chromosome of Clostridium botulinum type E strain Mashike. This fragment, cloned within plasmid pU9EMH, contains part of the structural gene for botulinum toxin type E neurotoxin as well as the entire structural gene for a nontoxic component of botulinum type E progenitor neurotoxin gene, ent-120. ent-120 is transcribed in the same direction as the neurotoxin gene and consists of one open reading frame encoding 1162 amino acid residues. Western blotting with anti-nontoxic component sera demonstrates that ent-120 encodes a protein of 120 kDa which forms part of the nontoxic component. ent-120 is homologous to an analogous gene found in botulinum type C strains (69.3% identity at the nucleotide level and 56.1% at the amino acid level). Two stretches of amino acids at the N-terminus of the ent-120 protein are highly homologous to amino acid sequences within the type E neurotoxin. The stop codon of the ent-120 gene is situated 27 nucleotides upstream from the start codon of the neurotoxin gene.
Assuntos
Toxinas Bacterianas , Toxinas Botulínicas/genética , Clostridium botulinum/genética , Genes Bacterianos/genética , Neurotoxinas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Western Blotting , Análise Mutacional de DNA , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Botulinum type D neurotoxin inhibited Ca(2+)-evoked norepinephrine secretion in digitonin permeabilized PC12 cells. Inhibition by the toxin required prior incubation with dithiothreitol (DTT). The inhibition was dependent on both concentration and incubation times of the toxin, and was affected by Ca2+ concentration. With less than 0.7 microM Ca2+ almost complete inhibition was observed; however, above 0.7 microM, Ca2+ stimulated additional norepinephrine release in a dose-dependent manner.
Assuntos
Toxinas Botulínicas/farmacologia , Digitonina/farmacologia , Norepinefrina/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Cálcio/metabolismo , Cálcio/fisiologia , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Células PC12 , Ésteres de Forbol/farmacologia , RatosRESUMO
Neurotoxins produced by Clostridium botulinum are classified into groups (A to G) based on their serological nature. They consist of two subunits, heavy and light chains, linked by one or more disulphide bridges. The light chain is responsible for the blocking of acetylcholine release. Amino acid sequences of light chains have already been reported for botulinum toxins types A, C, D and E. Five highly homologous regions are found between these four toxins. One of these homologous regions, sequence HELIHSL, shows strong similarity with the active site of zinc-proteases. We suggest that inhibition of acetylcholine release might be associated with this protease activity.
Assuntos
Toxinas Botulínicas/análise , Metaloendopeptidases/análise , Neurotoxinas/análise , Toxina Tetânica/análise , Acetilcolina/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Sinapses/enzimologiaRESUMO
The structural gene for a nontoxic-nonhemagglutinin component of Clostridium botulinum type C progenitor toxin was found to exist on a 7.8 kb DNA fragment obtained from a type C phage DNA. The gene existed between the neurotoxin and hemagglutinin genes, and consisted of an 3588 bp open reading frame (1196 amino acid residues). It was speculated that this gene and the neurotoxin gene were transcribed by the same mRNA (polycistronic transcription) in C. botulinum organisms.
Assuntos
Toxinas Botulínicas/genética , Clostridium botulinum/genética , Sequência de Aminoácidos , Sequência de Bases , Toxinas Botulínicas/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Substâncias Macromoleculares , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike. The 6.0 Kbp fragment coding for the entire light chain (L) component and the N-terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII. The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, C1, D, and tetanus toxins reported previously. The cleavage site of L and H components of type E toxin was presumed to be Arg-422. In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N-terminus of L component. On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.