| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1996: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
In 1995, Transposon mutagenesis of pPD1 with Tn917, Tn917lac, and Tn916 was carried out to generate insertion mutants which were transfer-deficient or make mating aggregates constitutively, to identify genes responsible for conjugal transfer and pheromone response. A part of DNA sequence of pheromone responding genes was determined.
In 1996, DNA sequence of all pheromone responding genes were determined and genes which had homology with pPD1 pheromone responding genes in amino acids sequence were searched in database. Pheromone responding genes of other pheromone responsive plasmids, pAD1 and pCF10. The order of pheromone responding genes of pPD1 was more similar to that of pCF10 than to that of pAD1.
All genes in the control region of pAD1 was studied well and their functions were getting clear. Although traA homologue in pCF10, prgX was found, it once had been thought to be a positive regulator while traA was negative regulator. An attempt was made to understand the function of traA gene in pPD1 by site-directed mutagenesis. An internal region of traA of pPD1 was amplified by PCR and cloned into E.coli vector with knanamycin resistance gene which could be expressed in E.faecalis. This plasmid was integrated into traA gene of pPD1 by homologous recombination resulting in inactivation of traA gene. This pPD1 with the insertion showed constitutive expresssion of conjugation system and high transfer frequency during short mating period. This result indicated that traA in pPD1 was also negative regulator as well as that of pAD1.
Summing up those results, three pheromone responsive plasmids with different characters were very similar in pheromone responding system, namely gene organization and their functions. In addition, it was quite likely that those plasmids had evolved from same origin.
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