, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2·5-7·0 kb inserts representing a 10 kb region of the genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30-170 kDa) in strains harbouring recombinant plasmids. As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300-600 kDa protein. This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A. No additional gene(s) with homology to the cloned toxin A gene was detected.


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