Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.     

Post‐chemiluminescence determination of chloramphenicol based on luminol–potassium periodate system

A post‐chemiluminescence (PCL) phenomenon was observed when chloramphenicol was injected into a mixture of luminol and potassium periodate after the chemiluminescence (CL) reaction of luminol–potassium periodate had finished. The possible reaction mechanism was proposed based on studies of the CL kinetic characteristics, the CL spectra, the fluorescence spectra and the UV‐vis absorption spectra of the related substances. Based on the PCL reaction, a rapid and sensitive method for the determination of chloramphenicol was established. The linear response range was 6.0 × 10^?7–1.0 × 10^?5 mol/L, with a correlation coefficient of 0.9986. The relative standard deviation (RSD) for 5.0 × 10^?6 mol/L chloramphenicol was 2.3% (n = 11). The detection limit was 1.6 × 10^?7 mol/L. The method has been applied to the determination of chloramphenicol in pharmaceutical samples with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

Overproduction of a mosquitocidal chloramphenicol-resistant Lysinibacillus sphaericus mutant obtained through UV irradiation

A highly active mosquitocidal mutant UV-chloramphenicol-resistant mutant (UCR-146) of Lysinibacillus sphaericus 2362 was isolated by UV irradiation and selected through resistance to chloramphenicol which was inhibitory for growth of the parent strain. The growth of UCR-146 in NYSMCL at different concentrations of chloramphenicol (20–100 µg/ml) showed high mosquitocidal activity against Culex pipiens larvae with the optimum concentration of 35µg/ml. At this level, LC₅₀ of UCR-146 was decreased about five times from that of L. sphaericus 2362. Comparative efficiency of mosquitocidal activity of UCR-146 and L. sphaericus 2362 within 28 days of consecutive growth exhibited notable stability of both cultures through seven cycles of growth in their optimal media. UCR-146 was grown in industrial by-products media for production of the binary toxin under economic conditions. Offal medium at 2% showed the highest mosquitocidal activity of UCR-146 with LC₅₀ of 0.53 PPM which was 16.6, 13, 12.3 and 3.4 times less than that produced with L. sphaericus 2362 grown in NYSM, L. sphaericus 2362 grown in offal, UCR-146 grown in NYSM and UCR-146 grown in NYSMCL, respectively. Hence, the mosquitocidal activity of L. sphaericus 2362 increased several times through ultraviolet (UV) irradiation followed by chloramphenicol resistance selection and then growth in 2% offal medium. Finally, this procedure for selecting UV-chloramphenicol-resistant mutant and the medium used could potentially be a simple and cost effective approach for obtaining and producing a highly active mosquitocidal mutant.     

Unstable, non-mutational expression of resistance to the thymidine analogue, trifluorothymidine in CHO cells.

In Saccharomyces cerevisiae, methyl methanesulphonate and diepoxybutane produced efficiently lethal, as well as mutagenic, damage in nuclear DNA. However, in the same conditions, these agents did not induce cytoplasmic petite mutations and poorly induced point mutations (resistance to erythromycin and chloramphenicol) in mitochondrial DNA. Possible reasons for these differences are discussed.     

Study of the maturation of 5 s RNA precursors in Escherichia coli

The existence of three precursors to 5 s RNA's (p5 s RNA's) has been confirmed. p5 s RNA's and mature 5 s RNA have different 5′-terminal sequences and produce the following 5′-terminal oligonucleotides: p5 s RNA-I:pAUUUG; p5 s RNA-II:pUUUG; p5 s RNA-III:pUUG; 5 s RNA:pUG. The results of experiments on pulse-labelled cells treated with actinomycin D, on chloramphenicol-inhibited cells, on various ribosome assembly-defective mutants and on the state of 5 s RNA in polysomes after a short labelling period, support the following conclusions. (1) p5 s RNA-I is the first identifiable precursor which appears during a pulse. (2) The amount of p5 s RNA-II, relative to those of the other p5 s RNA's, is very low at all times during pulse-chase experiments. On the contrary, it becomes significant during chloramphenicol inhibition and in one assembly-defective mutant under non-permissive conditions. (3) The maturation steps which lead to p5 s RNA-III and 5 s RNA normally occur after binding to 50 s subunit precursor particles and are, consequently, dependent upon protein synthesis. (4) The transition from p5 s RNA-III to 5 s RNA is a slow process, which is neither dependent upon nor required for the proper functioning of 50 s subunits in protein synthesis.     

Coupling of processing of alpha-glucosyl transferase mRNA with translation.

Bacteriophage T4 α-glucosyl transferase mRNA is made as a polycistronic 21S molecule that is processed during normal infection to the commonly found 14.5S species. By using antibiotic inhibitors of protein synthesis, it is possible to distinguish two steps involved in the processing of the 21S polycistronic α-gt mRNA in T4-infected $\text{Escherichia coli}$. There is an initial cleavage to an 18S molecule that does not require protein synthesis. However, the next step, the conversion of the 18S into the 14.5S molecule, requires simultaneous protein synthesis.