Affinity and hydrophobic chromatography of three variants of chloramphenicol acetyltransferases specified by R factors in <i>Escherichia coli</i>

Zaidenzaig, Yeshayahu; Shaw, William V.

doi:10.1016/0014-5793(76)80072-5

Cited by 49 publications

(27 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CAT was immunoprecipitated from each cleared extract according to Kessler (1976) using antiserum against purified CAT (Zaidenzaig & Shaw, 1976). An equal amount of extract protein from each sample was denatured and run on a 125 O/ O (w/v) SDS-discontinuous polyacrylamide gel (Laemmli, 1970).…”

Section: Methodsmentioning

confidence: 99%

Stationary phase, amino acid limitation and recovery from stationary phase modulate the stability and translation of chloramphenicol acetyltransferase mRNA and total mRNA in Escherichia coli

1998

View full text Add to dashboard Cite

The functional stability of the chloramphenicol acetyltransferase (cat) mRNA, as well as the functional stability of the total mRNA pool, change during the course of Escherichia coli culture growth. mRNA half-lives are long during lag phase, decrease during the exponential phase and increase again during the stationary phase of the bacterial growth cycle. The half-lives of cat mRNA and total mRNA also increase three-to fourfold during amino acid starvation when compared to exponential culture growth. Even though the stability of the cat message changes about fourfold during culture growth, the amount of cat mRNA per cell mass does not vary significantly between the culture growth phases, indicating that there are compensating changes in cat gene transcription. Translation of cat mRNA also changes during culture growth. In exponential phase, the rate of cat translation is about 14-fold higher than when the culture is in stationary phase. This is in contrast to the fourfold increase in stability of cat mRNA in the stationary-phase culture compared to the exponentially growing culture and indicates that active translation is not correlated with increased mRNA stability. When a stationary-phase culture was diluted into fresh medium, there was a five-to sevenfold increase in CAT synthesis and a threefold increase in total protein synthesis in the presence or absence of rifampicin. These results suggest that while mRNA becomes generally more stable and less translated in the stationary-phase culture, the mRNA is available for immediate translation when nutrients are provided to the culture even when transcription is inhibited.Of

show abstract

Section: Methodsmentioning

confidence: 99%

Stationary phase, amino acid limitation and recovery from stationary phase modulate the stability and translation of chloramphenicol acetyltransferase mRNA and total mRNA in Escherichia coli

1998

View full text Add to dashboard Cite

show abstract

“…Methods reported by Zaidenzaig and Shaw (34) and Packman and Shaw (25) were used to synthesize the Sephacryl-chloramphenicol (Sephacryl-CM) resin and to bind the fusion CAT to the resin (T. I. Zaghloul, Ph.D. thesis, University of California, Davis, 1986).…”

Section: Methodsmentioning

confidence: 99%

“…CAT activity was also determined by the rate of acetylation of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) the results with pGR71-43, pGR71, and no exogenous DNA were 0.95, 0.05, and 0 nmol of DTNB acetylated per min, respectively. Since CM is a substrate for CAT, the Sephacryl-CM resin binds CAT or CAT derivatives (25,28,34). When the reaction contained pGR71-43 as the template, a protein was synthesized that was capable of binding to the Sephacryl-CM resin.…”

Section: Methodsmentioning

confidence: 99%

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

Zaghloul¹,

Doi²

1987

J Bacteriol

View full text Add to dashboard Cite

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 4o29, 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.To study the regulation of gene expression in Bacillus subtilis, it is desirable to develop a coupled in vitro proteinsynthesizing system derived totally from this organism. The differences in promoters, transcriptional apparatus, transcriptional regulatory mechanisms (7), and translation signals and machinery (19,26,27,30,31) between grampositive and gram-negative organisms invalidate the use of the Escherichia coli or any other heterologous in vitro protein-synthesizing system in the analysis of gene expression in B. subtilis.Thus, although several in vitro protein-synthesizing systems have been described for several Bacillus species (3,6,13,18,20), these systems used mainly synthetic, endogenous, or exogenous RNA as the translational template. These systems also used several components or fractions derived from E. coli, which could not only complicate the interpretation of the results but also lead to faulty conclusions.To overcome these difficulties, we have developed a DNA-directed cell-free protein-synthesizing system that is derived totally from B. subtilis. This system contains readily prepared components, takes advantage of added RNA polymerase and tRNA prepared from B. subtilis, and is effective in terms of rate and specificity of synthesis.This system was able to synthesize an active enzyme by use of one of the transposon Tn9-derived chloramphenicol acetyltransferase (cat) gene fusions, pGR71-43, as the DNA template (9, 10). Furthermore, the product was characterized by its electrophoretic mobility on polyacrylamide gels, its binding characteristics to a specific affinity column, and its interaction with anti-CAT antiserum.MATERIALS AND METHODS Bacterial strains and media. B. subtilis 168 cells grown in superrich medium (11) were used to prepare the components for the in vitro system. E. coli HB101 cells carrying plasmid pBR328 were used to purify CAT (28).Preparation of nucleic acids. Plasmid pGR71 and its derivatives were purified by the procedure described by Lovett

show abstract

“…Active fractions were again pooled and concentrated with an Amicon Centriprep-10. A chloramphenicol caproate-agarose affinity column (Sigma) was used to remove a major contaminant, chloramphenicolacetyltransferase encoded by the vector pBR328 (64). OpdA was adsorbed in 10 mM Tris-HCl (pH 7.6) and eluted with 0.2 M NaCI.…”

mentioning

confidence: 99%

Cloning and nucleotide sequence of opdA, the gene encoding oligopeptidase A in Salmonella typhimurium

Conlin

Miller

1992

J Bacteriol

View full text Add to dashboard Cite

Affinity and hydrophobic chromatography of three variants of chloramphenicol acetyltransferases specified by R factors in Escherichia coli

Cited by 49 publications

References 17 publications

Stationary phase, amino acid limitation and recovery from stationary phase modulate the stability and translation of chloramphenicol acetyltransferase mRNA and total mRNA in Escherichia coli

Stationary phase, amino acid limitation and recovery from stationary phase modulate the stability and translation of chloramphenicol acetyltransferase mRNA and total mRNA in Escherichia coli

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

Cloning and nucleotide sequence of opdA, the gene encoding oligopeptidase A in Salmonella typhimurium

Contact Info

Product

Resources

About