In vitro expression of a Tn9-derived chloramphenicol acetyltransferase gene fusion by using a Bacillus subtilis system

A coupled in vitro protein-synthesizing system has been developed with components derived totally from Bacillus subtilis. The system synthesized specific gene products from various exogenous DNA templates, including B. subtilis phage phi 29, plasmid pUB110, and a heterologous B. subtilis-Escherichia coli gene fusion containing the transposon Tn9-derived chloramphenicol acetyltransferase (cat) gene. The gene fusion product was able to show CAT activity, bind specifically to a Sephacryl-chloramphenicol column, and react immunologically against anti-CAT antiserum. The fidelity of this in vitro system was demonstrated by the synthesis of gene products identical to that made in vivo. We suggest that this system may be used to study the regulation of gene expression in vitro.

[1]  R. Doi,et al.  A procedure to remove protease activities from Bacillus subtilis sporulating cells and their crude extracts. , 1977, Analytical biochemistry.

[2]  F. Kawamura,et al.  Translational coupling in Bacillus subtilis of a heterologous Bacillus subtilis-Escherichia coli gene fusion , 1985, Journal of bacteriology.

[3]  G. Chambliss,et al.  Natural Messenger Ribonucleic Acid-Directed Cell-Free Protein-Synthesizing System of Bacillus subtilis , 1974, Journal of bacteriology.

[4]  P. S. Lovett,et al.  Bacillus subtilis as a host for molecular cloning. , 1979, Methods in Enzymology.

[5]  L. Changchien,et al.  Spermidine Requirement for Bacillus thuringiensis Ribosomes in Cell-Free Phenylalanine Incorporation , 1970, Journal of bacteriology.

[6]  M. Stallcup,et al.  Initiation of protein synthesis in vitro by a clostridial system. I. Specificity in the translation of natural messenger ribonucleic acids. , 1973, The Journal of biological chemistry.

[7]  P. Cuatrecasas,et al.  A simplified method for cyanogen bromide activation of agarose for affinity chromatography. , 1974, Analytical biochemistry.

[8]  J. Rabinowitz,et al.  Specificity of promoter site utilization in vitro by bacterial RNA polymerases on Bacillus phage phi 29 DNA. Transcription mapping with exonuclease III. , 1980, Journal of Biological Chemistry.

[9]  R. Laskey.  The use of intensifying screens or organic scintillators for visualizing radioactive molecules resolved by gel electrophoresis. , 1980, Methods in enzymology.

[10]  D. Goldfarb,et al.  Translational block to expression of the Escherichia coli Tn9-derived chloramphenicol-resistance gene in Bacillus subtilis. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[11]  D. Dubnau,et al.  Studies on the synthesis of plasmid-coded proteins and their control in Bacillus subtilis minicells. , 1979, Plasmid.

[12]  G. Chambliss,et al.  DNA-directed cell-free protein-synthesizing system of Bacillus subtilis. , 1979, Biochimica et biophysica acta.

[13]  D. Goldfarb,et al.  Expression of Tn9-derived chloramphenicol resistance in Bacillus subtilis , 1981, Nature.

[14]  F. Kawamura,et al.  Analysis of ∅29 and ∅15 genomes by bacterial restriction endonucleases, EcoR1 and HpaI , 1976 .

[15]  R. Doi,et al.  Multiple procaryotic ribonucleic acid polymerase sigma factors. , 1986, Microbiological reviews.

[16]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[17]  W. V. Shaw.  Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. , 1975, Methods in enzymology.

[18]  J. Ito,et al.  DNA Replication of bacteriophage phi29: isolation of a DNA-protein complex from Bacillus subtilis cells infected with wild-type and with a suppressor-sensitive mutant. , 1976, Virology.

[19]  W. Bushuk,et al.  Purification of wheat proteases by affinity chromatography on hemoglobin-Sepharose column. , 1969, Biochemical and biophysical research communications.

[20]  W. V. Shaw,et al.  Affinity and hydrophobic chromatography of three variants of chloramphenicol acetyltransferases specified by R factors in Escherichia coli , 1976, FEBS letters.

[21]  M. Stallcup,et al.  Specificity of bacterial ribosomes and messenger ribonucleic acids in protein synthesis reactions in vitro. , 1976, Journal of Biological Chemistry.

[22]  J. Rabinowitz,et al.  Protein synthesis in Bacillus subtilis. I. Hydrodynamics and in vitro functional properties of ribosomes from B. subtilis W168. , 1979, Journal of molecular biology.

[23]  F. Kawamura,et al.  Bacteriophage gene expression in sporulating cells of Bacillus subtilis 168. , 1974, Virology.

[24]  J. Rabinowitz,et al.  Protein synthesis in Bacillus subtilis. II. Selective translation of natural mRNAs and its possible relation to the species-specific inhibition of protein synthesis by lincomycin and erythromycin. , 1979, Journal of molecular biology.

[25]  K. Burtis,et al.  Reconstitution studies show that rifampicin resistance is determined by the largest polypeptide of Bacillus subtilis RNA polymerase. , 1977, The Journal of biological chemistry.

[26]  J. Carrascosa,et al.  Synthesis in vitro of phi29-specific early proteins directed by phage DNA. , 1975, European journal of biochemistry.

[27]  W. Bonner,et al.  A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. , 1974, European journal of biochemistry.