1887

Abstract

A 1·4 kb positive regulatory element () that controls staphylococcal exfoliative toxin A (sETA) transcription was cloned from . is located upstream of the cloned 5·8 kb gene () obtained from the chomosomal DNA of ZM, the standard ETA-producing strain. The cETA prepared from an transformant into which the recombinant plasmid p (5·8 kb /pUC9) had been introduced was expressed at high levels in the culture supernatant and the ammonium-sulfate-precipitated culture supernatant fraction as shown by immunoblotting and the single radial immunodiffusion test. However, cETA produced by the recombinant plasmid p containing the 1·7 kb sequence () with a 1·45 kb -deficient fragment (1·7 kb /pUC9) obtained from the 5·8 kb sequence by subcloning was not detected in either the culture supernatant or the ammonium-sulfate-precipitated culture supernatant fraction (167-fold concentrate of the culture supernatant) by immunoblotting or the single radial immunodiffusion test. A large amount of cETA was produced by the 1·7 kb sequence when it was linked to amplified by PCR (1·7 kb -/pUC9), regardless of the orientation of insertion. Northern blot hybridization showed lower levels of the transcripts of the 1·7 kb sequence than of the 5·8 kb sequence. The rsETA prepared from an transformant into which the recombinant plasmid 3·4 kb -/pYT3 (pYT3-) had been introduced was expressed at high levels in the culture supernatant fraction as shown by the latex agglutination test. However, the agglutination titre in the culture supernatant fraction of rsETA produced by the recombinant plasmid (1·7 kb eta/pYT3) containing the 1·7 kb sequence carrying the 1·4 kb -deficient fragment (pYT3-) was 2500–4000 times lower than that of pYT3-.

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2004-04-01
2019-11-15
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