|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1990 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1989 : ¥1,600,000 (Direct Cost : ¥1,600,000)
1. Agrobacterium tumefaciens is a soil bacterium which induces crown gall tumors in a wide variety of dicotyledonous plants. This tumorigenic interaction between the bacterium and the plant is a natural example of the genetic engineering of eukaryotes. Indeed, the oncogenic bacterium harbors a large Ti plasmid, whose T-DNA region is transferred to and integrated into the plant genome during tumorous transformation. However, very little information is available concerning the early process leading to tumor formation. In order to study further the mechanism of tumor induction, it is necessary to develop a chemical probe that will help in exploring the early steps of tumor formation. We searched transformation inhibitors which do not inhibit the growth of A. tumefaciens and are nontoxic against the transformed plant cells.
2. A few esters derived from oxazolomycin isolated from Streptomyces sp. KBFP-2025 as well as two active compounds of bisanthraquinone type, julichrome Q_<1・3> and julim
ycin B-II, isolated from Streptomyces sp. TM-71, inhibit crown gall formation, but are nontoxic against this bacterium and the host plant.
3. These compounds administered to a potato disk 12 - 24 hours after the inoculation of the bacterium hardly inhibited crown gall formation, and no longer necrosed the potato disk. This suggests that these compounds act on any step of transformation process, but have no toxicity toward transformed plant cells whose genome has integrated the T-DNA of A. tumefaciens. It is probable that the transformation process of this system is completed within 12 - 24 hours after the inoculation. For a direct proof of this hypothesis, a quantitative microanalytical method of opines, indices of transformation, has to be established. This method involves HPLC analysis of fluorescent compound formed by reaction of benzoin with guanidino group of the characteristic amino acid. Time course of transformation will be possibly traced by this method.
3.ポテトディスクに本細菌を接種して,12〜24時間後に上記の化合物を投与したところ,形質転換の阻害がほとんど観察されず,正常なクラウンゴールが生成した。このことからこれらの化合物は形質転換のいずれかの段階に作用するものであり,形質転換した細胞には阻害を示さないことが判った。また,ポテトディスクの形質転換は 12〜24時間以内に終了していると推測されるが,それを直接証明するため形質転換の指標であるオパインの微量定量法を考案した。ベンゾインを,オクトピン,ノパリンのグアニジノ基と反応させ,蛍光物質 アミノー4,5ージフェニルイミダゾール誘導体とし,HPLCで分離定量する方法を確立した。この微量定量法により形質転換の時間経過を追跡することが可能となった。 Less