|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1991 : ¥300,000 (Direct Cost : ¥300,000)
Fiscal Year 1990 : ¥1,400,000 (Direct Cost : ¥1,400,000)
A. thaliana is most suitable for handling in the aspect of molecular generics to thoroughly Investigate mechanisms goveming the transcriptional regulation.
1. A Model Experiment for Screening Photosynthetic Mutants
A. thaliana grew on the agar medium of Gamborg B5 in supplementation with glucose or sucrose even in the presence of the inhibitor of photosynthetic electron transport, 3-(3, 4-dich ; orophenyQ-1, 1-dimethylurea(DCMU), although it did not grow without sugar sources under that condition. The resulting A. thaliana grown with inhibited level of photosynthetic activity, generated high red fluorescence upon exposure to a long wave UV light.
2. Mutagenesis of A thaliana transformed with Reporter and Suicide Gones
Genes for P-glucuronidase(GUS)and chloramphenicol acetyltransferase(CAT)as reporter genes, as well as a gene for alcohol dehydrogenase(ADH)as a suicide gene, were tried to introduce into A. thaliana prior to mutagenesis with ethylmethanesuffonate(EMS). This strategy may promi
se to make the selection of objective mutants easily in combination with screening by high red fluorescence. We have focused on members of genes for small subunit of ribulose 1, 5 -bisphosphate carboxylase/oxygenase(RuBisCO, rbcS-3B)and chlorophyll a/binding proteins(cabl). Polymerase chain reaction(PCR)with thermostable DNA polymerases associated with a 3'-5' proofreading exonuclease activity, was used to quickly obtain approximately 1.5-kilobase pair(kbp)DNA fragments covering promoters and their upstream regions of rbcS-38and cabl, as nucleotide sequences of these genes have been published. A gene for ADH(approximately 2.2 kbp)was similarly amplilied from A. thaliana DNA. The fragments possessing the promoter and regulatory activities of rbcS-3B and cabl were-placed upstream of genes for GUS, CAT, and ADH, and tried to introduce Into A. thaliana(Columbia and Bensheim, adhl. The transgenic A. thaliana is mutagenized with EMS, and mutants which less express rbcS-3B or cabl in green leaves is being screened. Less