| Research Abstract |
1. Of about 30-kb region of the virulence (vir) loci, essential for T-DNA transfer from bacteria to plant cells, on the hairy-root-inducing plasmid (pRiA4) from Agrobacterium rhizogenesare, a 17-kb region was sequenced. The overall organization and structure of most vir genes were similar to those of tumor-inducing plasmid pTiA6NC. In contrast, the spacers between vir genes were not conserved except for characteristic 6-bp sequences. Various mutations were introduced within the vir genes, and functions for several vir genes in the T-DNA transfer were deduced or estimated.
2. Transcription from the vir genes under the presence or absence of plant factors were analyzed by the Sl-mapping procedure, and it was found that (1) RNA start sites for each of vira and virg were different, depending on the conditions used, meaning the tandem presence of two promoters, which were constitutive and inducible, respectively; (2) one to four characteristic 6-bp sequences, phasing at ll-bp intervals, were present upstream all of the inducible promoters; (3) these 6-bp blocks were recognized by the VirG regulatory protein in vitro; (4) the phase of the 6-bp sequences was nearly opposite to that of the -35 and -10 regions of the promoter; and (5) based on these facts, we presented a model for transcriptional activation that VirG molecules cooperatively bind to DNA along one side by recognizing the 6-bp sequences, leading to the activation of the -35 region of promoter for RNA polymerase to interact with the DNA from the other side. 3. Plasmid vectors with a low-copy-number, which are useful for genetic analysis with Agrobacterium, were constructed by isolating mini-plasmids from pRiA4 and pTiB6S3.
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