| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1997: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
The gene coding for exfoliative toxin C (ETC) was cloned from the 42kb plasmid DNA of a Staphylococcus aureus strain into pUC119 and expressed in Escherichia coli strain JM105. The genes coding for exfoliative toxin B,C,D (shETB,shETC and shETD) were also cloned from the 42kb plasmids of Staphylococcus hyicus strains into pUC119 and expressed in E.coli strain JM105. Recombinant plasmids were recovered from ETC-, shETB-, shETC- and shETD-producing transformants and genes coding for ETC,shETB,shETC and shETD were shown to belocated within 2.1kb Xba l fragment, 1.7kb Hindlll fragment, 3.5kb Hindlll fragment 4.7kb Hindlll fragment, respectively. The gene coding for exfoliative toxin A (shETA) was cloned from the genomic DNA of a S.hyicus strain into pUC119 and expressed in E.colistrain JM105. Recombinant plasmid was recovered from a shETA-producing transformant and the gene coding for shETA was shown to be located within a 3.5kb Hindiii fragment. ETC,shETA,shETB,shETC and shETD produced by
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the E.coli transformants was indistinguishable from those produced by original hosts. Each recombinant toxin was not retained in bacterial cells and almost all toxins were extracellular. Nucleotide sequences of the genes coding for shETB and ETC (etc, heb) were determined by dideoxy method. Open reading frames (ORE) of the etc and heb genes were 804bp and 834bp, respectively. Shine- Dalgar no ribosome binding sites were detected at 8 to 10 bp upstr eams of their OREs. Promotor regions were also detected at upstreams of their ORFs. Translation of the ORFs of heb and etc genes yielded 268-and 278-amino-acid polypeptides. Twenty and 35 amino acid sequences downstr eams of the N-terminus of the shETB and ETC proteins were estimated as signal peptides. The conser vative sequences among genes coding for mshETB,ETA and ETB (Exfoliative toxin A and B produced by S.aureus) were presentedin the N-terminal portion of their molecules, while those among genes coding for ETC,ETA,ETB and shETB were not presented. Homologies of the estimated amino acid sequences between shETB and ETB and between shETB and ETA were appr oximately 60% and 40%. However, the homologies of the estimated amino acid sequences among ETC and other exfoliative toxins was only 20%. Less
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