DSRNA VIRUS-LIKE PLASMIDS IN YEAST

酵母中的 DSRNA 病毒样质粒

• 批准号: 3270155
• 负责人: DONALD J TIPPER
• 金额: $ 20.6万
• 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 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270155
• 关键词: RNA biosynthesis; Saccharomyces; autoradiography; cell differentiation; double stranded RNA; eukaryote; fungal genetics; gel electrophoresis; genetic manipulation; genetic strain; genetic transcription; genetic translation; immunochemistry; laboratory rabbit; microorganism toxin; mutant; nucleic acid denaturation; nucleic acid inhibitor; plasmids; protein structure; radiotracer; virus envelope; virus genetics; virus protein; 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 杀手M1-dsRNA最近被构建并被发现表达1型 在缺乏M1-dsRNA的细胞中，免疫和毒素水平较高。它的 表达将在转录和毒素水平上进行研究 体内的前体加工。该基因的修饰对小鼠胸腺功能的影响 将研究表达式以理解要求(例如， 糖基化)以有效地分泌、切割和发挥其功能 组成部分(先导Delta、毒素成分Alpha和Beta以及 推测的糖基化免疫决定簇GAMA DeltaAlphaGammaBeta)。 与特定肽下游的点进行框内融合的位点 PpTox序列中的裂解点(DeltaAlpha、AlphaGamma和 GammaBeta边界)被引入用于蛋白质的插入 序列以允许单独分析切割事件和检测 将pptox基因片段作为分泌信号用于外源毒素的分泌 酵母中的蛋白质。将与来自于 前α因子基因MFAlpha1和分泌酸基因PH05 磷酸酶。β-内酰胺酶将被用作一种强大的、易于检测的 将使用目标蛋白质和显色底物来允许 高分泌型酿酒酵母突变株的筛选。变种人将会是 分析特定处理事件的效率变化，并 确定分泌增强的阶段。 SKI5是一种次要的分泌型蛋白酶，它的基因将被克隆，并将其 所研究的产品。在与Keith Bostian博士的合作下， 将研究TPCK对ppTox加工的影响，克隆KEX1基因，并将其 可能涉及ppTox加工的产品将被分析。