| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1999: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Research Abstract |
Induction of the Taka-amylase A gene (taaG2) in A.nidulans may involve three steps ; generation of the strong ioducer isomaltose, signal transduction from isomaltose to the nuclear factor SREB that binds to the upstream activating sequence SRE, and transcriptional activation of taaG2 by SREB.We have studied on this model pathway and obtained the results as shown below.
1. Generation of isomaltose. A nidulans possessed an enzyme, IPE, that produced isomaltose from maltose. IPE was purified and its basic enzymatic properties were determined. The IPE gene was isolated and sequenced IPE contained amino acid sequences conserved among α -glucosidases. However, the overall homology was extremely low when compared to any known α-glucosedases, indicating that IPE is a new α- glucosidase.
2. DNA binding properties of the transcriptional activator AmyR.The gene encoding AmyR, which seemed to be identical to SREB, was cloned from A nidulans. AmyR possessed a Cys_6Zn (II) DNA binding motif at its N-t
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erminus. The recombinant AmyR produced in E.coli specifically bound to the promoter regions of the taaG2 and agdA (α-glucosidase) genes. The target sequence on the agdA promoter was CGGN_8CGG, and disruption of either one of the CGG triplets greatly reduced binding affinity of AmyR.On the other hand, the target sequence on the taaG2 promoter (CGGAAATT) displayed high binding affinity in spite that it lacked the down stream CGG triplet.
3. Functional domains of AmyR.Various C-terminal truncated AmyRs were constructed and analyzed for the abilities of DNA binding and transcriptional activation. The N-terminal DNA binding domain could bind to the cognate targets by itself. Truncation of the C-terminus up to the residue 413 resulted in a constitutive activator, indicating that an inhibitory domain against transactivation existed in this region.
In this research project, promoter analyses of the eglA gene encoding a cellulase were also carried out. Deletion of a specific region of the promoter lead to constitutive expression, suggesting that the gene is regulated by a traanscriptional repressor Less
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