RESUMEN
An 8 kb segment of the Clostridium botulinum NCTC 2916 genome 5' to the type A botulinum neurotoxin gene has been sequenced revealing five open reading frames. Four encode components (HA70, HA17, HA34 and NTNH/A) of the progenitor toxin complex. The product of the fifth, OrfX, possesses a putative C-terminal helix-turn-helix motif, exhibits homology with known regulatory proteins (including MsmR from Streptococcus mutans, UviA from C. perfringens and Orftxe1 located upstream of the C. difficile toxin B gene) and is also found within the vicinity of genes encoding tetanus toxin and types B, C, D and G botulinum toxins. Primer extension and Northern blotting analysis demonstrates that the genes are expressed as two divergent operons [HA34, HA17, HA70] and [NTNH/A, type A toxin gene], with the OrfX gene expressed singly. Immediately adjacent to the transcriptional start sites of the HA34 and NTNH/A genes are two highly conserved motifs (5'-ATTTTagGTTTACAAAA-3' and 5'-ATGTTATATgTaA-3'), separated by 12 bp, that span the putative -35 and -10 promoter regions. Homologous sequences occur in the equivalent position relative to the genes at type C botulinum toxin gene and the tetanus toxin gene loci. It is likely that these sequence motifs, together with OrfX, are involved in the co-ordinate expression of the genes encoding the various components of the botulinum toxin complex in groups I, III and IV C. botulinum strains and in that of the tetanus toxin gene.
Asunto(s)
Toxinas Botulínicas/genética , Clostridium botulinum/genética , Genes Bacterianos , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Bacteriano/genética , Genes Reguladores , Hemaglutininas/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas , Homología de Secuencia de AminoácidoRESUMEN
DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.
Asunto(s)
Toxinas Botulínicas/genética , Clostridium botulinum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Genes Bacterianos , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
The motions of objects in the environment reflect underlying dynamical constraints and regularities. The conditions under which people are sensitive to natural dynamics are considered. In particular, the article considers what determines whether observers can distinguish canonical and anomalous dynamics when viewing ongoing events. The extent to which such perceptual appreciations are integrated with and influence common-sense reasoning about mechanical events is examined. It is concluded that animation evokes accurate dynamical intuitions when there is only 1 dimension of information that is of dynamical relevance. This advantage is lost when the observed motion reflects higher dimension dynamics or when the kinematic information is removed or degraded.
Asunto(s)
Atención , Percepción de Forma , Percepción de Movimiento , Orientación , Física , Adulto , Formación de Concepto , Aprendizaje Discriminativo , Femenino , Humanos , Masculino , Fenómenos Físicos , Solución de Problemas , PsicofísicaRESUMEN
The entire structural gene of the Clostridium botulinum NCTC 11219 type-E neurotoxin (BoNT/E) has been cloned as five overlapping DNA fragments, generated by polymerase chain reaction (PCR). Analysis of triplicate clones of each fragment, derived from three independent PCR, has allowed the derivation of the entire nucleotide sequence of the BoNT/E gene. Translation of the sequence has shown BoNT/E to consist of 1252 amino acids and, as such, represents the smallest BoNT characterised to date. The light chain of the toxin exhibits the highest level of sequence similarity to tetanus toxin (TeTx, 40%). The light chains of BoNT/A and BoNT/D share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity. In contrast, the TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively. Comparisons with partial amino acid sequences of the light chain of BoNT/E from C. botulinum strain Beluga and that from the strains Mashike, Iwanai and Otaru, indicate single amino acid differences in each case. Alignment of all characterised neurotoxin sequences (BoNT/A, BoNT/C, BoNT/D, BoNT/E and TeTx) shows them to be composed of highly conserved amino acid domains interspersed with amino acid tracts exhibiting little overall similarity. The most divergent region corresponds to the extreme COOH-terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.
Asunto(s)
Toxinas Botulínicas/genética , Clostridium botulinum/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Bacterianos , Clonación Molecular , ADN Bacteriano/genética , Genes Bacterianos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Plásmidos , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
In five experiments, assessments were made of people's understandings about the dynamics of wheels. It was found that undergraduates make highly erroneous dynamical judgments about the motions of this commonplace event, both in explicit problem-solving contexts and when viewing ongoing events. These problems were also presented to bicycle racers and high-school physics teachers; both groups were found to exhibit misunderstandings similar to those of naive undergraduates. Findings were related to our account of dynamical event complexity. The essence of this account is that people encounter difficulties when evaluating the dynamics of any mechanical system that has more than one dynamically relevant object parameter. A rotating wheel is multidimensional in this respect: in addition to the motion of its center of mass, its mass distribution is also of dynamical relevance. People do not spontaneously form the essential multidimensional quantities required to adequately evaluate wheel dynamics.