ABSTRACT
The thermal dependencies of the apparent K(m) of the glutathione reductases from spinach (Spinacia oleracea L.) corn (Zea mays L.), and cucumber (Cucumis sativus L.) were determined. The apparent K(m) of the enzymes were found to vary up to 9-fold between 12.5 and 45 degrees C. Values of the apparent K(m) in excess of 200% of the observed minimum are suggested to be detrimental to the normal function of the enzyme. We propose the term "thermal kinetic window" to describe to the range of temperatures over which the apparent K(m) of the glutathione reductase is within 200% of its minimum and suggest that it may be a useful indicator of the limits of thermal stress for a given species. The thermal kinetic windows determined in this study are: <16 degrees C for spinach, 23 to 32 degrees C for corn, and 35 to 41 degrees C for cucumber.
ABSTRACT
A study of the use of carrier RNA to improve precipitation of DNA from dilute solutions was conducted to define the conditions which optimize DNA recovery. Replicate samples containing labeled pBR322 and increasing concentrations of commercially-available Torula yeast RNA were ethanol precipitated at -20 degrees C for 1 h in microfuge tubes obtained from various manufacturers. Nucleic acids were pelleted by centrifugation for either 5 or 30 min, dried and resuspended. Although recovery was not identical in each type of microfuge tube, in all cases the percent recovery increased when carrier was added. In most cases, extending centrifugation to 30 min did not significantly increase recovery. Recovery of unlabeled DNA's of heterogeneous molecular weight and conformation was also enhanced by the addition of carrier RNA. DNA's recovered by this method can be successfully digested with BamHI and ligated with T4 DNA ligase.
Subject(s)
DNA/isolation & purification , RNA , Chemical Precipitation , DNA, Bacterial/isolation & purification , DNA, Circular/isolation & purification , DNA, Superhelical/isolation & purification , DNA, Viral/isolation & purification , Plasmids , RNA, Fungal , SolutionsABSTRACT
A plasmid vector for cloning in Bacillus sphaericus 1593 was constructed in B. subtilis from two parent plasmids, pBC16 and pBD64. When characterized, the 3.9-MDa chimeric plasmid pNN101 was found to consist of the MspI fragment containing the chloramphenicol acetyltransferase (CAT) gene from pBD64 inserted into an MspI site in pBC16. pNN101 was shown to replicate, express, and be stably maintained in B. sphaericus 1593 without affecting the mosquito larvicidal activity of this organism. A derivative of this plasmid, pNN302, was constructed in which a unique HindIII site was introduced into the CAT gene without loss of chloramphenicol resistance.
