RESUMO
The cluster of genes encoding components of the progenitor botulinum neurotoxin complex has been mapped and cloned in Clostridium botulinum type G strain ATCC 27322. Determination of the nucleotide sequence of the region has revealed open reading frames encoding nontoxic components of the complex, upstream of the gene encoding BoNT/G (botG). The arrangement of these genes differs from that in strains of other antigenic toxin types. Immediately upstream of botG lies a gene encoding a protein of 1198 amino acids, which shows homology with the nontoxic-nonhemagglutinin (NTNH) component of the progenitor complex. Further upstream there are genes encoding proteins with homology to hemagglutinin components (HA-17, HA-70) and a putative positive regulator of gene expression (P-21). Sequence comparison has shown that BoNT/G has highest homology with BoNT/B. The sequence of the BoNT-cluster of genes in non-proteolytic C. botulinum type B strain Eklund 17B has been extended to include the complete NTNH and HA-17, and partial HA-70 gene sequences. Comparison of NTNH/G with other NTNHs reveals that it shows highest homology with NTNH/B consistent with the genealogical affinity shown between BoNT/G and BoNT/B genes.
Assuntos
Toxinas Botulínicas/genética , Clostridium botulinum/genética , Genes Bacterianos , Família Multigênica , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Clostridium botulinum/classificação , Primers do DNA/genética , DNA Bacteriano/genética , Evolução Molecular , Hemaglutininas/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
The neurotoxin gene from Clostridium botulinum type G was cloned as a series of overlapping DNA fragments generated using polymerase chain reaction (PCR) technology and primers designed to conserved regions of published botulinal toxin (BoNT) sequences. The 5'-end of the gene was obtained using a primer based on a conserved region of the nontoxic-nonhaemagglutinin gene lying upstream of the toxin gene. Translation of the nucleotide sequence derived from the cloned PCR fragments demonstrated that the gene encodes a protein of 1297 amino acid residues (rmm 149, 147). Comparative alignment of the determined BoNT/G sequence with those of other clostridial neurotoxins revealed highest sequence relatedness (approx. 58% amino acid identity) with BoNT/B of proteolytic and non-proteolytic C. botulinum. Tetanus toxin (TeTx) and other BoNT types revealed lower levels of relatedness with BoNT/G (approximate range 35-42% amino acid identity).