A GENETIC ANALYSIS OF MINIBRAIN KINASE IN C. ELEGANS

本研究计划的目标是确定在正常神经元功能和唐氏综合症认知缺陷中涉及小脑激酶（mnbK）的分子途径。对21号染色体部分三体的研究表明，相当一部分唐氏综合征表型来自21g22.2区域的2mb区域；mnbK定位于这个基因组区域。mnbK在小鼠中过表达会导致严重的学习和记忆缺陷，强烈暗示mnbK与唐氏综合征的认知缺陷有关。目前，mnbK的底物和调节因子尚不清楚。为了阐明这一途径，我们将研究秀丽隐杆线虫的mnbK同源物mbk-1和mbk-2。我建议表征mbk基因产物的表达和亚细胞定位，通过化学诱变结合群体PCR产生零等位基因，并通过过表达野生型和激活基因产物来模拟Down条件。作为确定MBK活性焦点的工具，四环素依赖的transactivator系统将适用于秀丽隐杆线虫，这是一个允许时间和空间调节基因表达的系统。将对候选基因进行测试，以确定它们是否属于mbk通路，并进行先导抑制子和/或增强子筛选，以公正的方式确定上游和下游成分。

The goal of this research proposal is to identify molecular pathways involving minibrain kinase (mnbK), both in normal neuronal function and in the cognitive defects of Down syndrome. Studies of partial trisomy of chromosome 21 suggest that a considerable portion of the Down syndrome phenotype result from a 2-Mb region at the 21g22.2 region; mnbK localizes to this genomic region. Overexpression of mnbK in mice causes severe defects in learning and memory, strongly implicating mnbK in the cognitive defects of Down syndrome. Currently, the substrates and regulators of mnbK are unknown. To elucidate this pathway, the Caenorhabditis elegans mnbK homologues mbk-1 and mbk-2, will be investigated. I propose to characterize the expression and subcellular localization of the mbk gene products, generate null alleles via chemical mutagenesis coupled with population PCR, and simulate the Down condition by overexpressing the wildtype and activated gene products. As a tool for determing the focus of MBK activity, the tetracycline-dependent transactivator system will be adapted to C. elegans, a system that permits temporally and spatially regulated gene expression. Candidate genes will be tested to determine if they belong to a mbk pathway, and pilot suppressor and/or enhancer screens will be performed to identify upstream and downstream components in an unbiased manner.