DSRNA VIRUS-LIKE PLASMIDS IN YEAST

酵母中的 DSRNA 病毒样质粒

• 批准号: 3270152
• 负责人: DONALD J TIPPER
• 金额: $ 19.5万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270152
• 关键词: Saccharomyces; autoradiography; cell differentiation; double stranded RNA; eukaryote; fungal genetics; gel electrophoresis; genetic manipulation; genetic strain; genetic transcription; genetic translation; immunochemistry; microorganism toxin; mutant; nucleic acid denaturation; nucleic acid inhibitor; plasmids; radiotracer; virus envelope; virus genetics; virus replication; viruslike particle; yeasts

A complete cDNA copy of the preprotoxin (ppTox, M1-P1) gene of type 1 killer M1-dsRNA has recently been constructed and found to express type 1 immunity and toxin at high levels in cells lacking M1-dsRNA. Its expression will be studied at the levels of transcription and toxin precursor processing in vivo. The effects of modification of this gene on expression will be studied in order to understand the requirements (e.g., glycosylation) for efficient secretion, cleavage and function of its component parts (leader Delta, toxin components Alpha and Beta and presumptive glycosylated immunity determinant Gamma in the order DeltaAlphaGammaBeta). Sites for in frame fusion to points just downstream of specific peptide cleavage points in the ppTox sequence (the DeltaAlpha, AlphaGamma and GammaBeta boundaries) are being introduced for insertion of protein sequences to allow individual assay of the cleavage events and testing of the pptox gene fragments as secretion signals for the secretion of foreign proteins in yeast. Comparisons will be made with signals derived from the MFAlpha1 gene for prepro Alpha Factor and the PH05 gene for secreted acid phosphatase. Beta-lactamase will be employed as a robust, easily assayed target protein and chromogenic substrates will be employed to allow screening for hypersecreting S. cerevisiae mutants. Mutants will be analyzed for changes in the efficiency of specific processing events and to identify the stage of secretion enhanced. The gene for SKI5, a minor secreted protease, will be cloned, and its product studied. In collaboration with Dr. Keith Bostian, the effects of TPCK on ppTox processing will be studied, the KEX1 gene cloned, and its product, probably involved in ppTox processing, will be analyzed.

1型前原毒素（ppTox，M1-P1）基因的完整cDNA拷贝 最近构建了杀伤M1-dsRNA，发现其表达1型 在缺乏M1-dsRNA的细胞中具有高水平的免疫力和毒素。 其 表达将在转录和毒素水平上进行研究 体内前体加工。 这种基因的修饰对 将研究表达式以便理解要求（例如， 糖基化）以有效分泌、切割和发挥其功能 组成部分（先导Delta，毒素组分Alpha和Beta， 假定的糖基化免疫决定簇γ， DeltaAlphaGammaBeta）。 与特定肽下游点的框内融合位点 ppTox序列中的切割点（DeltaAlpha、AlphaGamma和 GammaBeta边界）用于插入蛋白质 序列，以允许单独测定切割事件和检测 PPTOX基因片段作为分泌信号用于分泌外源蛋白， 酵母中的蛋白质 将与来自以下的信号进行比较： 前原α因子的MF α 1基因和分泌酸的PH 05基因 磷酸酶。 β-内酰胺酶将作为一种耐用、易于测定的 将使用靶蛋白和显色底物 筛选分泌过多的S.酿酒酵母突变体。 变种人将会 分析特定处理事件的效率变化， 确定分泌增强的阶段。 SKI 5是一种分泌性蛋白酶，将被克隆， 产品研究 在与基思博斯蒂安博士的合作中， 将研究TPCK对ppTox加工的影响，克隆KEX 1基因，并对其进行鉴定。 将分析可能涉及ppTox处理的产品。