RANDOM MUTATION ANALYSIS OF THE PRP GENE

遗传学研究表明PrP基因突变可能与 这种疾病在受感染动物身上的病程。也许最令人兴奋的是它 似乎PrP ORF中的基因变化也可能促进其 从头开始。本研究旨在探讨PrP在蛋白质合成中的作用。 瘙痒病的突变分析。第一个目标是建设 随机突变的仓鼠和小鼠PrP基因库。目标明确 随机诱变将用于生产突变仓鼠和 小鼠PrP基因。ORF将被划分为任意域和一个 将为多肽的每一部分构建单独的文库 编码区。所使用的ORF盒式磁带将被概括性地标记为 允许区分外源重组蛋白和内源重组蛋白 细胞PrP。将使用pSPOXI.neo载体来表达这些突变体 培养中感染和未感染的小鼠和仓鼠细胞中的基因。 我们将采用两种截然不同的策略。首先是一名代表 变种人的目录将被组装起来。这些突变体将被描述为 通过测序来确定突变，并将在体内测试 脂质体介导的人皮肤瘙痒病特异活性研究 培养中感染或未感染瘙痒病的小鼠和仓鼠细胞。在……里面 添加随机突变的仓鼠和小鼠ORF的池将是 在感染和未感染小鼠N2a和叙利亚仓鼠SH中表达 细胞。影响PrP对话资格的突变 对蛋白水解酶具有抗性的突变株，以及影响 异源基因控制的PrPSc“阻断”活性将被鉴定 并通过DNA测序来定义。这些化验将在 突变PrP基因的代表性横截面。此外，提供 可以开发一种合适的原位菌落分析，随机池 从文库中挑选出来的将通过原位免疫分析进行筛选 菌落复制到聚酯或硝化纤维滤网上。在……里面 此外，我们将筛选能够启动从头开始的突变。 在培养细胞中出现PrPSc和/或感染性普恩病毒 筛选出了。这些化验也将在两个人身上进行， 描述克隆以及随机克隆池的特征。此外, 将对部分纯化的转染体提取物进行生物检测 细胞。

Genetic studies indicate that mutations in the PrP gene may contribute to the course of the disease in infected animals. Perhaps most excitingly it appears that genetic changes in the PrP ORF may also perhaps promote its de novo appearance. This study intends to examine the role of PrP in scrapie by mutational analysis. The first objective is the construction of libraries of randomly mutated hamster and mouse PrP genes. Targeted random mutagenesis will be used to produce libraries of mutant hamster and mouse PrP genes. The ORFs will be divided into arbitrary domains and a separate library will be constructed for each section of the polypeptide coding region. The ORF cassettes used will be epitopically "tagged" to allow discrimination of the exogenous recombinant protein from endogenous cellular PrP. The pSPOXII.neo vector will be used to express these mutant genes in both infected and uninfected murine and hamster cells in culture. Two distinct strategies will be employed. Firstly a representative catalog of mutants will be assembled. These mutants will be characterized by sequencing to define the mutation and will be tested in vivo for scrapie-specific activity following lipofectin mediated transfection into scrapie-infected or uninfected mouse and hamster cells in culture. In addition pools of randomly mutated hamster and mouse ORFs will be expressed in both infected and uninfected mouse N2a and Syrian hamster SH cells. Mutations which affect the eligibility of PrP for conversation to the protease-resistant for, as well as mutants that affect the heterologous gene directed PrPSc "blocking" activity will be identified and defined by DNA sequencing. These assays will be performed on a representative cross-section of mutant PrP genes. In addition, providing that a suitable in situ colony assay can be developed, pools of randomly selected from the library will be screened by in situ immunoassay of colonies replicated onto polyester or nitrocellulose filters. In addition, we shall screen for mutations which can initiate the de novo appearance of PrPSc and/or infectious prions in cultured cells will be screened for. These assays will also be performed on both individual, characterized clones as well as on pools of random clones. In addition, bioassays will be performed on partially purified extracts of transfectant cells.