CLONING AND FUNCTIONAL CHARACTERIZATION OF INHERITED NEURODEGENERATIVE DISORDERS

这一项目需要识别与一种 人类遗传性疾病的种类。这些措施包括发展和 改进基因作图和分离的方法，以及 为了更好地研究这些基因的特性和功能分析 对其致病机制的了解将导致治疗 和干预。使用位置克隆策略，我们的实验室 参与了亨廷顿病(HD)基因的鉴定和 共济失调-毛细血管扩张基因(A-T)。HD是一个进步的， 中年起病的神经退行性疾病。而该基因包含 一种多态(CAG)n三核苷酸重复序列，当 重复次数扩展到42次或更多。基因的亚细胞表达和定位 全长cDNA结构的正常和扩展版本为 在杆状病毒和哺乳动物表达系统中进行检测。在……里面 此外，正在开发小鼠YAC转基因株系 显微注射含有完整HD基因的YAC 将扩增的CAG重复序列改造到受精的原核中 鸡蛋。A-T是一种常染色体隐性遗传病，其特征是 进行性小脑变性，眼皮肤毛细血管扩张， 免疫缺陷、辐射敏感性、癌症易感性和 染色体不稳定。目前对A-T的研究工作涉及到 同源序列在葡萄球菌中的作用；抗血清的制备 A-T蛋白的特异性；表达模式的特征 和蛋白质产物的定位；以及基因敲除小鼠的产生 模特们。当前的位置克隆项目涉及识别 Niemann-Pick型C型的缺陷基因。 常染色体隐性遗传性神经内脏脂肪储存障碍 核上型突出的患者的神经功能减退 凝视麻痹，典型伴随进行性认知 功能障碍、共济失调、肌张力障碍、吞咽困难、构音障碍、癫痫发作、猝倒 和痴呆症。重组研究指出了18q11-q12的定位， 更具体地说，在标记D18S44和D18S480之间的2 cM间隔内。 我们目前正在通过搜索来完善基因图谱 有关其他多态标记和组装YAC和粘粒重叠群的信息 对于该地区来说。候选基因正在通过cdna选择和 外显子陷阱。此外，整个YAC正在被引入NPC 细胞，以检查细胞缺陷的纠正。利用 在NPC小鼠模型中，正在进行回交以进一步 确定小鼠的致病部位并确定合并区 在人类身上。一种新的基因鉴定方法也在进行中， 这涉及到YAC片段载体的开发，该载体包括3‘ 外显子捕捉盒，酵母可选标记，Alu序列 靶向，酵母端粒序列。中心版本和中心版本 载体是用Alu序列制成的，这两个序列也存在于 方向。

This project entails the identification of genes responsible for a variety of human inherited disorders. These include development and refinement of methodologies for gene mapping and isolation, and the characterization and functional analyses of these genes for a better understanding of their pathogenic mechanisms that will lead to therapy and intervention. Using positional cloning strategies, our laboratory participated in the identification the Huntington Disease (HD) gene and the ataxia-telangiectasia gene (A-T). HD is a progressive, neurodegenerative disorder with onset in midlife. and the gene contains a polymorphic (CAG)n trinucleotide repeat which is pathogenic when the repeats expand to 42 or more. Subcellular expression and localization of the normal and expanded versions of the full length cDNA constructs are being examined in baculovirus and mammalian expression systems. In addition, mouse YAC transgenic lines are being developed by microinjecting a YAC containing the entire HD gene that has been retrofitted with an expanded CAG repeat into the pronucleus of fertilized eggs. A-T is an autosomal recessive disorder characterized by progressive cerebellar degeneration, oculocutaneous telangiectasia, immunodeficiency, radiosensitivity, susceptibility to cancer and chromosomal instability. Current research efforts for A-T involves the function of homologous sequences in S. cerevisae; generation of antisera specific for the A-T protein; characterization of the expression pattern and localization of the protein product; and generation of knockout mouse models. A current positional cloning project involves the identification of the defective gene for Niemann-Pick type C. NPC is an autosomal-recessive, neurovisceral lipid storage disorder marked by neurological decline in patients which is highlighted by supranuclear gaze palsy and is typically accompanied by progressive cognitive impairment, ataxia, dystonia, dysphagia, dysarthria, seizures, cataplexy and dementia. Recombinant studies point to a localization at 18q11-q12, more specifically in a 2 cM interval between markers D18S44 and D18S480. We are currently in the process of refining the genetic map by searching for additional polymorphic markers and assembling YAC and cosmid contigs for the region. Candidate genes are being isolated by cDNA selection and exon trapping. Additionally, entire YACs are being introduced into NPC cells to check for correction of the cellular defect. Taking advantage of an NPC mouse model, backcrosses are being performed to further delimeate the affected locus in mice and identify the synthenic region in humans. A novel method for gene identification is also underway and this involves development of a YAC fragmentation vector that includes 3' exon trapping cassette, yeast selectable marker, Alu sequences for targeting, yeast telomere sequences. Acentric and centric versions on the vector are being made with Alu sequences also found in both orientations.