Analyses of signal transduction system involved in induction of polysaccharide degrading enzymes in filamentous fangi

在构巢曲霉中，Taka-amylase A基因（taaG 2）的诱导可能包括三个步骤：产生强碘诱导剂异麦芽糖，从异麦芽糖到与上游激活序列SRE结合的核因子SREB的信号转导，以及SREB对taaG 2的转录激活.异麦芽糖的生成。一个nidulans拥有一种酶，IPE，生产异麦芽糖从麦芽糖。对IPE进行了纯化，并测定了其基本酶学性质。分离了IPE基因并测序，IPE含有α -葡萄糖苷酶中保守的氨基酸序列。然而，与任何已知的α-葡萄糖苷酶相比，其整体同源性极低，表明IPE是一种新的α-葡萄糖苷酶.转录激活因子AmyR的DNA结合特性。编码AmyR的基因似乎与SREB相同，从构巢曲霉中克隆。AmyR在其N端具有Cys_6Zn（II）DNA结合基序 ...更多信息 厄米纳斯在大肠杆菌中产生的重组AmyR特异性结合到taaG 2和agdA（α-葡萄糖苷酶）基因的启动子区。agdA启动子上的靶序列为CGGN_8CGG，破坏CGG三联体中的任一个都大大降低了AmyR的结合亲和力。另一方面，taaG 2启动子上的靶序列（CGGAAATT）尽管缺乏下游CGG三联体，但仍显示出高结合亲和力。AmyR的功能结构域：构建了各种C端截短的AmyR，并分析了其与DNA结合和转录激活的能力。N-末端DNA结合结构域可以通过自身与同源靶标结合。C-末端截短至413位残基导致组成型激活因子，表明该区域存在反式激活抑制结构域。启动子特定区域的缺失导致组成型表达，表明该基因受转录抑制因子的调控。

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 … More 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