TnphoA: a transposon probe for protein export signals.
Proceedings of the National Academy of SciencesPublished 1 December 1985Open access
Colin Manoil, J Beckwith
Citations1,020
Generate an AI Snapshot to get a quick, structured summary of this paper.
Study Snapshot
ObjectiveStudy objective
MethodsResearch methodology
PopulationPopulation studied
Sample sizeSample sizes
OutcomesStudy outcomes here
ResultsStudy results comes here
LimitationsResearch study limitations comes here
A concise AI-generated summary of the paper will appear here once you click Generate AI Snapshot.
TL;DR
A derivative of transposon Tn5 is constructed that permits the generation of hybrid proteins composed of alkaline phosphatase lacking its signal peptide fused to amino-terminal sequences of other proteins, and should help localize export signals within the structure of a protein, such as a transmembrane protein, as well as identify new chromosomal genes for secreted and trans Membrane proteins.
Abstract
We constructed a derivative of transposon Tn5 that permits the generation of hybrid proteins composed of alkaline phosphatase (EC 3.1.3.1) lacking its signal peptide fused to amino-terminal sequences of other proteins. Such a hybrid gives alkaline phosphatase activity if the protein fused to alkaline phosphatase contributes sequences that promote export and thus compensate for the missing alkaline phosphatase signal peptide. Fusions to both a secreted periplasmic protein and a complex cytoplasmic membrane protein led to alkaline phosphatase activity. TnphoA fusions should help localize export signals within the structure of a protein, such as a transmembrane protein, as well as identify new chromosomal genes for secreted and transmembrane proteins.
Keywords
MedicineBiochemistry, Genetics and Molecular Biology
NatureCleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
251,640 Citations1970Ulrich K. Laemmli
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Cellular Physiology and BiochemistryShort-Term Effects of Nose-Only Cigarette Smoke Exposure on Glutathione Redox Homeostasis, Cytochrome P450 1A1/2 and Respiratory Enzyme Activities in Mice Tissues
47,283 Citations2013Haider Raza, Annie John +1 more
The results suggest that even short term exposure of cigarette smoke have adverse effects on mitochondrial functions and redox homeostasis in tissues which may progress to further complications associated with chronic smoking.
Cold Spring Harbor Symposia on Quantitative BiologyComplete Nucleotide Sequence of the Escherichia coli Plasmid pBR322
1,775 Citations1979J G Sutcliffe
GeneThe use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids — a review
420 Citations1984Frans J. de Bruijn, James R. Lupski
The properties of transposon Tn5 that render it useful for in vivo mutagenesis of cloned DNA sequences are reviewed and a methods section is included which outlines precisely how to carry out transposition frequency, insertional specificity, polarity and stability of Tn 5 insertion mutations.
CellProtein localization in E. coli: Is there a common step in the secretion of periplasmic and outer-membrane proteins?
360 Citations1981Koreaki Ito, P J Bassford +1 more
The results suggest that most of the periplasmic and outer-membrane proteins share a common step in localization before the polypeptide becomes accessible to the processing enzyme.
CellSecretion of beta-lactamase requires the carboxy end of the protein
314 Citations1980Douglas Koshland, David Botstein
Results show that the carboxy-terminal amino acid sequence of beta-lactamase is essential to successful transport across the cytoplasmic membrane, and suggest that the presence (and probably also the act of removal) of the signal sequence does not suffice to ensure secretion.
Journal of BacteriologyMutations that alter the signal sequence of alkaline phosphatase in Escherichia coli
280 Citations1983S Michaelis, Hiroshi Inouye +2 more
Two mutations lie within the phoA portion of the fused gene and cause internalization of the hybrid protein and lead to amino acid alterations in the signal sequence of alkaline phosphatase.
Journal of BacteriologyUse of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm
265 Citations1979P J Bassford, Thomas J. Silhavy +1 more
The results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites, and indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.
Proceedings of the National Academy of SciencesFusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion.
245 Citations1985Charles S. Hoffman, Andrew Wright
The ability to change the amino terminus of phosphatase without altering its activity makes the enzyme particularly useful for construction of protein fusions.
Cold Spring Harbor Symposia on Quantitative BiologyStructural Analysis of Tn5
238 Citations1981Ennes A. Auerswald, G. Ludwig +1 more
The results support the view that the inverted repeats of Tn5 stem from two identical copies of an originally independently moving DNA element, and seem to have evolved toward a close physical and functional linkage with the antibiotic-resistance gene.
Journal of Biological ChemistryPrecise location of two promoters for the beta-lactamase gene of pBR322. S1 mapping of ribonucleic acid isolated from Escherichia coli or synthesized in vitro.
211 Citations1982J. Brosius, Richard L. Cate +1 more
S1 mapping of procaryotic mRNA, transcribed in vivo, allows both an accurate identification of promoters and the analysis of their transcriptional regulation.
GeneThe plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication
207 Citations1981Pierre Prentki, François Karch +2 more
The plasmid pBR325 carries between the Cmr and Tcr genes a 482 bp sequence which duplicates, in the opposite orientation, a section pf pBR322 located at the end of the tcr gene.
Proceedings of the National Academy of SciencesConstruction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus.
188 Citations1984Lee Kroos, D Kaiser
Tn5 lac could be used to assay transcription in any bacterium in which Tn5 can transpose and beta-galactosidase can be measured, and on the regulation of the promoter of the transcription unit, verifying that by transposition Tn 5 lac can fuse lacZ expression to outside promoters.
CellEvidence for posttranslational translocation of β-lactamase across the bacterial inner membrane
153 Citations1982Douglas Koshland, David Botstein
Cold Spring Harbor Symposia on Quantitative BiologyTransposition of the Lac Region of E. coli
142 Citations1966J Beckwith, Ethan R. Signer +1 more
Proceedings of the National Academy of SciencesCellular location affects protein stability in Escherichia coli.
141 Citations1982Karen Talmadge, W Gilbert
A full-length preproinsulin precursor appears transiently before the signal sequence is cleaved off by the bacterial signal peptidase, and a model is proposed to account for this.
Journal of Biological ChemistryThe role of the beta-lactamase signal sequence in the secretion of proteins by Escherichia coli.
130 Citations1984James T. Kadonaga, Antoine Gautier +4 more
The view that there are characteristics of the mature protein that are necessary for the secretion across the inner membrane of E. coli is supported.
GeneSequence homology between the tetracycline-resistance determinants of Tn10 and pBR322
119 Citations1983Toai Nguyen, Kathleen Postle +1 more
It is concluded that a single Tn10 gene, the tetA gene, is sufficient to confer tetracycline resistance and that a substantial portion of these proteins may be embedded within the cytoplasmic membrane.
GeneticsSPECIFICITY OF TRANSPOSON Tn<i>5</i> INSERTION
108 Citations1983Douglas E. Berg, Margaret Schmandt +1 more
DNA sequence analysis indicated that GC base pairs occupy positions 1 and 9 in the duplications at each of the five hotspots examined, suggesting a GC-cutting preference during Tn5 transposition.
CellRegulation of Tn5 by the right-repeat proteins: Control at the level of the transposition reaction?
98 Citations1982Ralph R. Isberg, Aili L. Lazaar +1 more
By assaying expression of proteins that are hybrids between beta-galactosidase and IS50R proteins, it is found that the regulation of transposition cannot be due to the inhibitor repressing synthesis of Tn5 proteins.
Journal of BacteriologySignal sequence of alkaline phosphatase of Escherichia coli
97 Citations1982Hiroshi Inouye, W. S. Barnes +1 more
The alteration of DNA sequence was identified for a promoter mutation that results in the expression of phoA independent of the positive control gene phoB and in insensitivity to high phosphate.
Journal of BacteriologyMating aggregates in Escherichia coli conjugation
97 Citations1975Mark Achtman
A comparison of light microscopy, sucrose gradient centrifugation, and analysis with a modified Coulter counter indicated that the number of cells in mating aggregates is best equantitated using amodified Coulter counter.
Journal of Molecular BiologyReplicon fusions promoted by the inverted repeats of Tn5
68 Citations1981Ralph R. Isberg, Michael Syvanen
It is shown that both repeats have the sites necessary for replicon fusions, but only the right stem has the required protein(s) and establishes that the right repeat is an insertion sequence.
Methods in enzymology on CD-ROM/Methods in enzymology[1] Genetic analysis of protein export in Escherichia coli
68 Citations1983Jonathan Beckwith, Thomas J. Silhavy
The cloning of the structural gene for an enzyme that cleaves signal sequences from precursors of secreted proteins will allow the genetic characterization of this locus and its function.
Proceedings of the National Academy of SciencesGeneration of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus.
48 Citations1984Vivian Bellofatto, Lucy Shapiro +1 more
Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences.
Journal of Biological ChemistryRenaturation and identification of periplasmic proteins in two-dimensional gels of Escherichia coli.
36 Citations1982Bruce R. Copeland, Rebecca J. Richter +1 more
A technique was developed for renaturation of the ligand binding sites of periplasmic binding proteins in denaturing two-dimensional gels to demonstrate that leucine-specific and cystine binding proteins both have different isoelectric points in different strains.
Journal of BacteriologyDeletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA
31 Citations1981Aparna V. Sarthy, S Michaelis +1 more
Lambda transducing phages containing portions of the phoA gene have been isolated and used to construct a deletion map of the PHOA gene.