| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Porphyromonas gingivalis (P.g.) has been generally thought that its transformation by plasmid DNA is impossible. The reason for it was elucidated in this study to be attributable to the presence of restriction enzyme (s) in this species, because transformation was possible when it was tried using plasmid DNAs extracted from the P.g. straine itself. Upon this knowlege, we then tried to obtain a mutant that lacks the restriction enzyme, usable as a good recipient strain for transformation experiments. For this, trials of transformation of strains that had been mutagenized with N-methyle-N'-nitro-N-nitrosoguanidin (NTG) with pE5-2 DNA were performed, and the transformants (erythromycin resistant colonies) thus obtained were cultured in the absence of the antibiotic to allow the plasmid segregation. By this means some strains that exhibited a capacity of incorporating foreign DNAs could be obtained. Subsequently, we made further efforts to find plasmids that can replicate and be stably maintained in P.g. cells. The only known plasmid pE5-2 that can be transferred into P.g. cells by either conjugation or transformation is extremely unstable in this species ; we presume that Bacteroides eggerthii, in which the rep gene of pE5-2 originated, is rather distant from P.g. A rep gene from a plasmid of a closer species would likely increase the stability. In this respect, we employed several recombinant plasmids that were constructed by ligating fragments of the plasmids detected from the black-pigmented oral anaerobic species and the erythromycin-resistnce fragment of pE5-2. Among them, we could successfuly found a plasmid, pYH400, a recombinant with the rep gene of a plasmid from Porphyromonas asaccharolytica, which could be maintained in the P.g.cells very stably. It would possibly be usable as a good cloning vector for this species.
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