GENETIC APPROACHES TO ANALYZING THE INSERTION & TOPOLOGY

分析插入的遗传学方法

• 批准号: 3484881
• 负责人: JONATHAN BECKWITH
• 金额: $ 15.95万
• 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 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3484881
• 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的方法来研究拓扑 整合膜蛋白的排列。 碱熔 磷酸酶（AP）与这些蛋白质的周质结构域的结合， 高AP酶活性，而AP与细胞质 结构域产生低的酶活性。 通过分离大量的 这种融合的整合膜蛋白，MalF和 前导肽酶，我们希望得到这些拓扑结构 膜蛋白 β-半乳糖苷酶（betaGz）与 膜蛋白的周质区表现出低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--情况正好匡威 在AP。 betaGz与膜蛋白融合的分析 还应提供拓扑信息。 一个β-Gz与MalF的融合物具有低β-Gz活性， 据推测，由于其膜插入，将用于获得 并表征了改变MalF插入的能力的突变体， 进入细胞膜。 该菌株的Lac+衍生物的选择 应该会产生malF基因的突变， 突变体将被表征，并应产生以下信息：1） MalF中负责其膜插入的信号和2） 任何参与插入过程的细胞成分。