Detergents inhibit chloramphenicol acetyl transferase.

Potent inhibition of chloramphenicol acetyl transferase (CAT) by Triton X-100 and Nonidet P-40 was observed. The CAT activity was also moderately inhibited by sodium deoxycholate and sodium dodecyl sulfate, and least by Tween 20. Detergents should, therefore, not be used for cell lysate preparation when CAT activity is used as the reporter in a transient expression experiment.     

Downstream sequence adjacent to AUG affects translation of chloramphenicol acetyl transferase in eukaryotic cells

The CAT gene is widely used as a reporter in eukaryotic systems because of the efficient translation of its mRNA. We report here that a sequence occurring in the CAT mRNA at +15 nucleotides from CAT AUG is essential for translation. This sequence includes a stem-loop structure, which, however, exhibits a calculated stability significantly lower than that required for a hairpin to act as an enhancer of translation in vitro. Replacement of this region with the corresponding sequence from mRNAs that are normally translated in eukaryotic systems drastically reduced translation of CAT in COS cells, although the consensus sequence around the AUG, known to be required for high-level translation initiation, was conserved. These observations may be relevant for the exploitation of the CAT reporter system for analysis of the mechanisms of translation initiation by means of fusion constructs.     

Quantitation of chloramphenicol acetyl transferase (CAT) messenger RNA by filter hybridisation using a digoxigenin label

Summary Slot- and dot-blotting are commonly used to evaluate levels of messenger ribonucleic acid (mRNA). Quantitation of bacterially-expressed chloramphenicol acetyl transferase (CAT) mRNA by this method is highly dependent on total RNA immobilised onto the solid support as well as mRNA concentration. mRNA quantitation by comparison with a pure standard results in underestimation. An improved protocol for CAT mRNA detection is described.     

Release of chloramphenicol acetyl transferase from recombinant Escherichia coli by sonication and the French press

Summary The release of chloramphenicol acetyl transferase (CAT) from a recombinant Escherichia coli strain by ultrasonication and the French press was compared. French pressing disrupted all cells in suspension whereas only a fraction of the cells was disrupted following sonication. The level of CAT released was highest when cells were totally disrupted. Additional treatment with the detergent Triton X-100 was necessary to maximize CAT recovery, presumably due to association of CAT with cellular debris.     

Liposome-mediated introduction of the chloramphenicol acetyl transferase (CAT) gene and its expression in tobacco protoplasts

The expression plasmid vector pUC8CaMVCAT, containing the chloramphenicol acetyl transferase (CAT) gene, was encapsulated in large unilamellar vesicles (LUV) and introduced into tobacco protoplasts derived from either cell suspension culture or leaf mesophyll. Treatment with liposomes took place in a buffer containing either NaCl or CaCl₂, but no polyethylene glycol. The presence of polylysine in the incubation buffer increased the adsorption of liposomes to protoplasts but decreased the efficiency of CAT gene expression.The expression of the introduced CAT gene could be monitored for at least seven days, following the treatment (about 25% acetylation at day 3 as well as at day 7). Plasmid DNA sequences could be detected, apparently unmodified, for at least nine days in the plant cells, though unintegrated in the host genome.     

Quantitation of chloramphenicol acetyl transferase in transgenic tobacco plants by ELISA and correlation with gene copy number

A monoclonal antibody to chloramphenicol acetyl transferase (CAT) was used in an indirect competitive enzyme immunoassay (ELISA) for the quantitation of CAT in leaf extracts of eighteen transgenic tobacco plants containing the CAT gene fused to the cauliflower mosaic virus 35S promoter. The ELISA could be used to quantify CAT when present in extracts at 20 ng/ml. Enzymatic activity and electrophoretic mobility of CAT in these extracts was not different from CAT from Escherichia coli. Concentrations of CAT in these transgenic plants ranged from 79 to 732 ng CAT/mg protein. The average coefficient of variation among three replicate samples was 15%. All plants were sampled on two separate occasions. The CAT concentrations often varied between the two sampling dates. We determined the CAT gene copy number and the number of independently segregating loci in each plant by Southern blot analysis and progeny testing. We found no significant differences in CAT expression among all ten plants with a single CAT gene. We also found a significant correlation between CAT gene copy number and the level of CAT expressed in each plant, although plants with one gene copy sometimes had more CAT than plants with more than one gene copy. In this population, therefore, gene copy number contributed more to the variation in CAT expression than did position effects.     

Assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus

Spodoptera frugiperda insect cells were grown in Sf-900 serum-free medium and two kinds of serum-supplemented media (IPL -41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h(-1) respectively, at 33 degrees C. The IPL -41 medium supported to highest maximum cell density (10.6 x 10(6) cells/mL) compared to 3.5 x 10(6) and 8.7 x 10(6) cells/mL with the Grace's and serum-free media, respectively. In temperature shifdown experiments with a temperature-sensitive baculo-virus (acts10YM1CAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL -41 (5.1 x 10(7) PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum-free medium (4.4 x 10(6) vs 4.1 x 10(5) PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02 At MOI of 2.0 virtually no increase in virus of CAT titer was observed. This result is contrary to those obtained at constant-temperature (27 degrees C) infection and cell culture, in which higher virus titers and recombinant protein expression and obtained at higher MOI.     

High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.

Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA.     

Chloramphenicol acetyl transferase activity in Brassica spp.

Upon discovery that Brassica campestris leaf extracts harbour some chloramphenicol acetyl transferase (CAT) activity, a systematic screening of plant tissue for this activity, so far only reported for prokaryotic microorganism, has been conducted. Results were negative for three solanaceous plants as well as for the Cruciferae Arabidopsis thaliana and Orychophragmus violaceus. By contrast, the three tested species of the Cruciferae genus Brassica exhibit significant CAT activity. The Brassica CAT activity is much more heat labile than the enzyme encoded by the bacterial transposon, Tn9, that is commonly used as a reporter in gene fusion experiments.