GENETIC APPROACHES TO ANALYZING THE INSERTION & TOPOLOGY

分析插入的遗传学方法

• 批准号: 3484883
• 负责人: JONATHAN BECKWITH
• 金额: $ 23.72万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3484883
• 关键词: Escherichia coli; alkaline phosphatase; bacterial genetics; bacterial proteins; beta galactosidase; chromosome deletion; cytoplasm; enzyme mechanism; exopeptidase; genetic manipulation; genetic mapping; membrane permeability; membrane proteins; plasmids; protein engineering; protein sequence; tissue /cell culture

We will use a gene fusion approach to study the topological arrangement of integral membrane proteins. Fusions of alkaline phosphatase (AP) to periplasmic domains of such proteins give high AP enzymatic activity, while fusions of AP to cytoplasmic domains give low enzymatic activity. By isolating a large number of such fusions to the integral membrane proteins, MalF and leader peptidase, we hope to derive the topology of these membrane proteins. Fusions of betagalactosidase (betaGz) to periplasmic domains of membrane proteins exhibit low beta-Gz activity, while fusions of betaGz to cytoplasmic domains of the same protein give high activity - the converse of the situation with AP. Analysis of betaGz fusions to membrane proteins should also give information on topology. One betaGz fusion to MalF which has low betaGz activity, presumably due to its membrane insertion, will be used to obtain and characterize mutants which alter the ability of MalF to insert into the membrane. Selection of Lac+ derivatives of this strain should yield both mutations in the malF gene and unlinked mutants will be characterized and should yield information on 1) the signals in MalF responsible for its membrane insertion and 2) any cellular components that are involved in the insertion process.

我们将使用基因融合方法来研究拓扑 完整膜蛋白的排列。 碱性融合物 磷酸酶（AP）连接到此类蛋白质的周质结构域 AP 酶活性高，同时 AP 与细胞质融合 结构域的酶活性较低。 通过隔离大量 这种与整合膜蛋白、MalF 和 前导肽酶，我们希望推导出这些的拓扑结构 膜蛋白。 β半乳糖苷酶 (betaGz) 的融合 膜蛋白的周质结构域表现出低β-Gz 活性，而 betaGz 与细胞质结构域的融合 相同的蛋白质具有高活性 - 情况相反 与美联社。 betaGz 与膜蛋白融合的分析 还应提供有关拓扑的信息。 MalF 的一种 betaGz 融合物具有低 betaGz 活性， 大概由于其膜插入，将被用来获得 并表征改变 MalF 插入能力的突变体 进入膜。 该菌株 Lac+ 衍生物的选择 应该在 malF 基因中产生突变并且未连锁 突变体将被表征并应产生有关 1) 的信息 MalF 中负责其膜插入的信号和 2) 插入过程中涉及的任何细胞成分。