The effect of changes in the bulk dielectric constant on the DNA torsional properties.

The effect of changes in the bulk dielectric constant on the DNA torsional properties was evaluated from plasmid circularization reactions. In these reactions, pUC18 previously linearized by EcoRI digestion was recircularized with T4 DNA ligase. The bulk dielectric constant of the reaction medium was decreased by the addition of different concentrations of neutral solutes: ethylene glycol, glycerol, sorbitol, and sucrose, or increased by the addition of glycine. The topoisomers generated by the ligase reaction were resolved by agarose-gel electrophoresis. The DNA twist energy parameter (kappa), which is an apparent torsional constant, was determined by linearization of the Gaussian topoisomers' distribution. It was observed that the twist energy parameter for the given solutes is almost linearly dependent on the bulk dielectric constant. In the reaction buffer, the twist energy parameter was determined to be 1100 +/- 100. By decreasing the dielectric constant to 74 with the addition of sorbitol, the value of the parameter reaches kappa = 900 +/- 100, whereas the addition of ethylene glycol leads to kappa = 400 +/- 50. Upon addition of glycine, which resulted in a dielectric constant equal to 91, the value of the twist energy parameter increased to kappa = 1750 +/- 100.

Identification of a progenitor of the CTX-M-9 group of extended-spectrum beta-lactamases from Kluyvera georgiana isolated in Guyana.

Chromosomal beta-lactamase genes (bla(KLUY)) from six Kluyvera georgiana strains isolated in Guyana were cloned and expressed in Escherichia coli. KLUY-1 exhibited 100% amino acid identity with the extended-spectrum beta-lactamase CTX-M-14. We also show that a 2.7-kb Kluyvera chromosomal region exhibits 99% nucleotide identity to a portion of In60 that includes bla(CTX-M-9).

Detection of genomic variability in different populations of the cattle tick Boophilus microplus in southern Brazil.

DNA from seven isolates of the cattle tick Boophilus microplus was analyzed by restriction fragment length polymorphism (RFLP). Three different cDNA clones, named P-9, P-25 and CP-12, isolated from a B. microplus cDNA library, were used as DNA probes. DNA sequences of P-9 have high similarity to ribosomal genes, whereas P-25 does not show significant homology with known sequences within databases. CP-12 is a cDNA clone encoding a cysteine endopeptidase gene. A limited degree of polymorphism was detected with P-9 and P-25, while CP-12 showed a different pattern of bands for each tick isolate. These findings suggest the existence of a complex genotypic diversity of the tick B. microplus population in endemic regions.

Stabilization of the restriction enzyme EcoRI dried with trehalose and other selected glass-forming solutes.

The stabilization of the restriction enzyme EcoRI by its incorporation into aqueous glass-forming carbohydrate or polymer solutions, followed by vacuum-drying to low moisture, has been studied. Glass-forming solutes included trehalose, sucrose, lactose, maltose, raffinose, maltodextrin DE 10, and poly(vinylpyrrolidone) (molecular weight 40,000, PVP). Among the solutes examined, trehalose and sucrose protected the enzyme most effectively during storage at 37 and 45 degrees C. The restriction enzyme dried with trehalose or sucrose maintained its activity without detectable loss for at least 20 days at 37 degrees C and 12 days at 45 degrees C. In contrast, the activity of the enzyme dried with maltodextrin or PVP was reduced during vacuum desiccation and also it decreased remarkably during storage at the same temperatures. Stored (37/45 degrees C) vacuum-dried trehalose and sucrose systems were either a dense paste or a very viscous syrup, and this indicated that they were not glassy. Moreover, no relationship was found between the glass transition temperatures (Tg) of the pure added solute and enzyme protection during storage, since, e.g., sucrose which has significantly lower Tg values protected the enzyme much better than either maltose, lactose, maltodextrin, or PVP. The trisaccharide raffinose offered good protection of enzyme activity, and its role as a novel excipient matrix for labile enzyme stabilization deserves further investigation. The stability of enzyme EcoRI was rapidly lost when the vacuum-dried trehalose and sucrose systems were humidified to 58% relative humidity and stored at 45 degrees C, and this was attributed to disaccharide crystallization.