ABSTRACT
A simple, fast and sensitive method to perform Southern transfer and hybridization with nonradioactive detection of genetic loci is described. With a 10-ng sample of human genomic DNA, alleles of the D2S44 locus can be detected within 7 h of completing gel electrophoresis.
Subject(s)
Blotting, Southern/methods , DNA/analysis , Luminescent Measurements , Cell Line , Deoxyribonucleases, Type II Site-Specific , Humans , Nucleic Acid Denaturation , Nucleic Acid Hybridization/methods , Ultraviolet RaysABSTRACT
We have followed the synthesis and secretion of a number of periplasmic and outer membrane proteins in three strains of Escherichia coli, a secA amber mutant, a secA temperature-sensitive mutant, and a strain that blocks protein secretion due to a high level of expression of an export-defective hybrid protein between maltose-binding protein and beta-galactosidase (MalE-LacZ). Our results show that after several hours under nonpermissive conditions the specificity and extent of the export blocks in the secA temperature-sensitive mutant and the strain producing the MalE-LacZ hybrid protein are identical, affecting at least four major outer membrane proteins and most but not all periplasmic proteins. The secA gene product, therefore, appears to be an essential component of the major export pathway in E. coli which is used by many envelope proteins independent of whether they are cotranslationally or post-translationally secreted. In contrast, the synthesis of only a subset of these envelope proteins is reduced in the secA amber mutant after shift to the nonpermissive condition. These results indicate that the SecA protein serves roles both in the synthesis and the secretion of certain cell envelope proteins.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/metabolism , Bacterial Outer Membrane Proteins/biosynthesis , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/physiology , Biological Transport , Electrophoresis, Polyacrylamide Gel , Escherichia coli/physiology , Protein Processing, Post-Translational , Recombinant Proteins/metabolismABSTRACT
Plasmids have been constructed in which the Escherichia coli alkaline phosphatase promoter and signal sequence have been fused to the staphylococcal nuclease gene to promote the high-level expression and secretion of this gene product in E. coli. We determined that the first amino acid residue after the signal sequence can determine whether this protein was processed and exported to the periplasmic space. Fractionation and protease accessibility studies were used to show that the export-defective, nuclease precursor is internal to the cytoplasmic membrane barrier of the cell. Furthermore, this export defect was suppressed in a strain containing a prlA mutation. These findings are novel in that this region of the polypeptide chain has been implicated in processing but not export and that prlA mutations have not been previously known to suppress such defects.
Subject(s)
Enzyme Precursors/metabolism , Genes, Bacterial , Micrococcal Nuclease/metabolism , Staphylococcus aureus/genetics , Alkaline Phosphatase/genetics , Cell Membrane/enzymology , DNA, Recombinant , Enzyme Precursors/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Micrococcal Nuclease/genetics , Mutation , Promoter Regions, Genetic , Protein Sorting Signals/metabolism , Staphylococcus aureus/enzymologyABSTRACT
We studied the dependence of prlA-mediated suppression of signal sequence mutations in maltose-binding protein on cellular SecA levels in Escherichia coli. Reduction of SecA levels within the cell had strong positive and negative effects on prlA-mediated suppression, depending on the particular signal sequence mutations involved. This finding suggests that prlA and secA gene products are both components of a common export system.