Genetics of Protein Secretionin Escherichia coli

将对大肠杆菌中的分泌机制进行研究，该机制涉及将蛋白质输出到细胞膜。各种遗传选择和筛选程序，以分离在该机器部件上有缺陷的突变体。这些突变体将定义SEC基因，这些基因将被克隆、测序并鉴定其基因产物。SEC基因产物的功能将通过在体外系统中检测SEC突变提取物的体内表型和行为来研究。将制定新的遴选程序。在一种方法中，对SecA-LacZ融合基因的去抑制选择会在已知的sec基因和新基因secE中产生突变。在第二程序中，在转变到指定的非允许温度之后，对不能将噬菌体lambda受体输出到外膜的选择进行。此外，将寻求允许在转变到不允许的温度后快速关闭分泌机械的程序和突变体。突变选择将被用来定义蛋白B-半乳糖苷酶的成分，这些成分阻止它通过细胞膜。这些研究可能有助于阐明蛋白质通过膜的机制或蛋白质折叠途径。这个项目涉及蛋白质跨膜运输(分泌)的机制。对高等生物的研究导致了信号假说的形成，在该假说中，一个不带电荷的疏水肽(信号序列)的存在促进了跨膜分泌。随后在细菌蛋白质上确定了信号序列，从而允许使用细菌遗传学的强大方法来分析分泌机制。将使用各种选择程序来分离分泌缺陷的细菌突变株。这些突变体将定义参与分泌过程的细菌基因，并将允许分离和鉴定这些基因的产物。该项目完成后，将提供细菌中蛋白质分泌的详细图片，并将作为高等生物体中类似研究的模型。

A study will be made of the secretion machinery in E. coli, involved in the export of proteins to the cell envelope. Various genetic selections and screening procedures to isolate mutants defective in components of this machinery. The mutants will define sec genes which will be cloned, sequenced and their gene products identified. The function of the sec gene products will be studied by examining in vivo phenotypes and behavior of sec mutant extracts in an in vitro system. New selection procedures will be developed. In one, a selection for derepression of a secA-lacZ fusion yields mutations in already known sec genes and in a new gene, secE. In a second procedure, selection for the inability to export bacteriophage lambda receptor to the outer membrane is carried out after a shift to a designated non-permissive temperature. In addition, procedures and mutants will be sought that allow a rapid shut-off of the secretion machinery after a shift to non-permissive temperature. Mutant selections will be used to define the components of the protein B-galactosidase that prevent it from passing through the cytoplasmic membrane. These studies may shed light either on the mechanism of protein passage through membranes or on protein folding pathways. This project is concerned with the mechanism by which proteins are transported (secreted) across membranes. Studies on higher organisms have led to formulation of the signal hypothesis in which the presence of an uncharged hydrophobic peptide (the signal sequence) facilitates transmembrane secretion. Signal sequences have subsequently been identified on bacterial proteins, thus allowing the powerful methods of bacterial genetics to be used to analyze the secretion machinery. A variety of selection procedures will be used to isolate bacterial mutants that are defective in secretion. These mutants will define the bacterial genes that participate in the secretion process and will allow isolation and characterization of the products of these genes. Upon completion, this project will provide a detailed picture of protein secretion in bacteria and will serve as a model for similar studies in higher organisms.