RANDOM MUTATION ANALYSIS OF THE PRP GENE

遗传学研究表明，PrP基因的突变可能有助于 受感染动物的病程。 也许最令人兴奋的是， 似乎PrP ORF中的遗传变化也可能促进其表达。 重新出现。 本研究旨在研究PrP在以下方面的作用： 通过突变分析确定羊瘙痒症。 第一个目标是建设 随机突变的仓鼠和小鼠PrP基因库。 针对性 随机诱变将用于产生突变仓鼠文库， 小鼠PrP基因。 ORF将被划分为任意域和一个 将为多肽的每个区段构建单独的文库 编码区 使用的ORF盒将被表位“标记”， 允许区分外源重组蛋白和内源重组蛋白 细胞PrP pSPOXII.neo载体将用于表达这些突变体 感染和未感染的鼠和仓鼠细胞中的基因。 将采用两种不同的战略。 首先是代表 变种人的目录将被收集。 这些突变体将被描述为 通过测序来确定突变，并将在体内进行测试， 脂质体介导转染后羊瘙痒症特异性活性 培养的羊瘙痒病感染或未感染的小鼠和仓鼠细胞。 在 将随机突变的仓鼠和小鼠ORF的添加池 在感染和未感染的小鼠N2 a和叙利亚仓鼠SH中表达 细胞 影响PrP转换资格的突变， 抗蛋白酶的森林，以及影响 将鉴定异源基因指导的PrPSc“阻断”活性 并通过DNA测序来确定。 这些试验将在 突变型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测定法， 将通过原位免疫测定筛选从文库中选择的 菌落复制到聚酯或硝化纤维滤膜上。 在 此外，我们将筛选可以启动从头突变的突变， 培养细胞中PrPSc和/或感染性朊病毒的出现将是 筛选为。 这些试验也将在两个个体上进行， 表征的克隆以及随机克隆的池。 此外，本发明还提供了一种方法， 将对部分纯化的转染子提取物进行生物测定 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.