No abstract

A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli a-S5 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.

The sequence of 20 amino acids from the N terminus of Clostridium perfringens epsilon-toxin was determined. Some differences between this sequence and the previously published sequence (A. S. Bhown and A. F. S. A. Habeeb, Biochem. Biophys. Res. Commun. 78:889-896, 1977) were found. A degenerate 23-bp pair oligonucleotide probe was designed from the amino acid sequence data and used to isolate a DNA fragment containing the gene encoding epsilon-toxin (etx) from C. perfringens type B. The gene encoded a protein with a molecular weight of 32,981. Upstream of the gene, promoter sequences which resembled the Escherichia coli a70 consensus sequences were identified. The gene was expressed in E. coli, and the cloned gene product reacted with epsilon-toxin-specific monoclonal antibodies and had a molecular weight and isoelectric point similar to those of the native protein. Downstream of etx, two overlapping open reading frames were identified. Each encoded part of a protein which was homologous to the transposase from Staphylococcus aureus transposon Tn4001. Southern hybridization experiments indicated that the etx gene was found only in C. perfringens types B and D, the types which produce epsilon-toxin.

The N-terminal domain of Clostridium perfringens alpha-toxin, homologous with the nontoxic phospholipase C of BaciUus cereus, was expressed in Escherichia coli and shown to retain all of the phosphatidylcholine hydrolyzing activity of the alpha-toxin, but not the sphingomyelinase, hemolytic, or lethal activities. The C-terminal domain of alpha-toxin showed sequence and predicted structural homologies with the N-terminal region of arachidonate 5-lipoxygenase, an enzyme from the human arachidonic acid pathway which plays a role in inflammatory and cardiovascular diseases in humans.

SUMMARYThe genome DNAs from the Panolis flammea (Pf) multiple nucleocapsid nuclear polyhedrosis virus (MNPV) and the Mamestra brassicae (Mb) MNPV were analysed with the restriction endonucleases BamHI, BgllI, KpnI, HindlII, Sinai and XhoI. The profiles produced by each enzyme for the two virus genomes were quite dissimilar with very few comigrating fragments. The size of PfMNPV DNA was calculated to be 145 kilobase pairs (kbp) and that of MbMNPV 150 kbp. Physical maps of the two genomes were constructed utilizing the above enzymes. The two maps were oriented in relation to their putative polyhedrin genes. Alignment of the two restriction maps for PfMNPV and MbMNPV was achieved by performing cross blot hybridization between XhoI digests of the two virus genomes. This showed that despite differences in the physical maps the two genomes shared overall similarity in gene organization. The two viruses were also compared using dot blot hybridization analysis to quantify homology with Autographa californica (AC) MNPV. These data showed that both PfMNPV and MbMNPV were distantly related to AcMNPV but exhibited a high degree of homology to each other (nearly 100% in 20% formamide, 70% in 50% formamide).