A plasmid vector for simultaneous generation of lacZ protein fusions and npt-II operon fusions in vivo

We have constructed a plasmid vector, pHLH1, that contains lacZ and npt-II genes flanked by ends of bacteriophage Mu. These sequences can be integrated into Escherichia coli chromosome by the Mu transposition system. The lacZ gene, deleted for its transcription and translation initiation signals, was positioned next to the 117 bp terminal fragment for the s end of Mu. The npt-II gene, deleted for its transcriptional signals, was positioned distal to lacZ in the same orientation. Transposition of this sequence to a target gene can create simultaneously a protein fusion to lacZ and an operon fusion to npt-II. To demonstrate the use of this vector, we isolated lacZnpt-II+ fusions to ompC. These fusions exhibited the expected phenotypes and regulational properties of ompC fusions.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  M. Van Montagu,et al.  Identification of plant promoters in situ by T‐DNA‐mediated transcriptional fusions to the npt‐II gene , 1986, The EMBO journal.

[3]  J. Beckwith,et al.  TnphoA: a transposon probe for protein export signals. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Beckwith,et al.  Uses of lac fusions for the study of biological problems. , 1985, Microbiological reviews.

[5]  E. Hurt,et al.  The first twelve amino acids (less than half of the pre‐sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. , 1985, The EMBO journal.

[6]  G. Weinstock,et al.  Transposable lambda placMu bacteriophages for creating lacZ operon fusions and kanamycin resistance insertions in Escherichia coli , 1985, Journal of bacteriology.

[7]  G. An,et al.  A Tn3 lacZ transposon for the random generation of beta‐galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. , 1985, The EMBO journal.

[8]  J. Way,et al.  New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. , 1984, Gene.

[9]  R. Sprengel,et al.  Protein fusions with the kanamycin resistance gene from transposon Tn5. , 1984, EMBO Journal.

[10]  J. Roth,et al.  Conditionally transposition-defective derivative of Mu d1(Amp Lac) , 1984, Journal of bacteriology.

[11]  G. Weinstock,et al.  Lambda placMu: a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step , 1984, Journal of bacteriology.

[12]  R. Bressan,et al.  Mutagenesis of Erwinia carotovora subsp. carotovora with bacteriophage Mu d1 (Apr lac cts62): construction of his-lac gene fusions , 1984, Journal of bacteriology.

[13]  E. Groisman,et al.  In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[14]  L. Shapiro,et al.  Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[15]  L. Siminovitch,et al.  Expression of Bacterial β-Galactosidase in Animal Cells , 1982 .

[16]  J. Ingraham,et al.  Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions , 1981, Journal of bacteriology.

[17]  D. Macneil,et al.  Regulation of nitrogen fixation in Klebsiella pneumoniae: isolation and characterization of strains with nif-lac fusions , 1981 .

[18]  G. Wilcox,et al.  Isolation of ara-lac gene fusions in Salmonella typhimurium LT2 by using transducing bacteriophage Mu d (Apr lac) , 1980, Journal of bacteriology.

[19]  J. Beckwith,et al.  Labeling of proteins with beta-galactosidase by gene fusion. Identification of a cytoplasmic membrane component of the Escherichia coli maltose transport system. , 1980, The Journal of biological chemistry.

[20]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[21]  M. Hall,et al.  Transcriptional regulation of Escherichia coli K-12 major outer membrane protein 1b , 1979, Journal of bacteriology.

[22]  S. Cohen,et al.  Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[23]  P. Reeves,et al.  Outer membrane proteins of Escherichia coli. VI. Protein alteration in bacteriophage-resistant mutants , 1977, Journal of bacteriology.

[24]  B. Allet,et al.  Partial correlation of the genetic and physical maps of bacteriophage Mu. , 1977, Virology.

[25]  M. Casadaban,et al.  Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. , 1976, Journal of molecular biology.

[26]  M. Montagu,et al.  Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase , 1985, Nature.

[27]  E. T. Palva,et al.  In vivo transfer of chromosomal mutations onto multicopy plasmids utilizing polA strains: cloning of an ompR2 mutation in Escherichia coli K-12 , 1985 .

[28]  T. Silhavy,et al.  Genetic analysis of protein export in Escherichia coli K12. , 1985, Annual review of biochemistry.

[29]  M. Casadaban,et al.  In vivo formation of gene fusions encoding hybrid beta-galactosidase proteins in one step with a transposable Mu-lac transducing phage. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .