Effect of glutaraldehyde on the activity of some DNA restriction endonucleases

The effect of the bifunctional crosslinking reagent glutaraldehyde on the activity of the restriction enzymes Bam HI,Hind III, EcoRI, and Tthlll I was investigated. The four enzymes exhibited differential sensitivity to inactivation. Tthlll I was the most sensitive, with activity losses occurring at levels of 0.0025% and above.Hind III was the most stable of the four and remained fully active at concentrations as high as 0.075%. Addition of BSA to incubation mixtures generally had a stabilizing effect. Implications of these results for the design of glutaraldehyde-based methods for the immobilization of restriction endonucleases are discussed.     

Reasons for the high stability of fumarase activity ofBrevibacterium flavum cells immobilized with k-carrageenan gel

Whole cells ofBrevibacterium flavum having high fumarase activity were immobilized using K-carrageenan. The reason for the high stability of fumarase activity of immobilized cells was investigated. One of main reasons for stabilizing fumarase activity by immobilization using K-carrageenan against organic solvents such as ethanol and acetone was the lower concentration of these solvents in the carrageenan gel compared with that in outer bulk solution. The stabilization of fumarase activity in the immobilized cells against protein-denaturing reagents was found to be related to rheological properties of K-carrageenan gel. Another reason for stabilizing fumarase activity by immobilization with K-carrageenan was to protect the cells from lysis. When immobilized cells were freeze-thawed, their fumarase activity increased and operation stability decreased. Therefore, one reason for the high decay of fumarase activity caused by the freeze-thawing may be a change in the pore size of the K-carrageenan gel. Fumarase activity and the operational stability of immobilized cells was found to depend on gelling conditions. Therefore, the steric structure of the K-carrageenan gel may be related to the decay of fumarase activity.     

Molecular assessment of S1 endonuclease-resistant snapback hairpin loops generated by DNA polymerase I during the in-vitro nick translation reaction

The in-vitro nick translation reaction used to label DNA to high specific activity also produces aberrant DNA structures known as “snapback” hairpin loops. Hairpin structures are precluded from participating in precise DNA-DNA hybridization interactions. Three nick translation systems were all found to yield significant quantities of snapback hairpins, as determined by their resistance to S1 endonuclease digestion following denaturation. The relative quantities of hairpins produced correlated with both the mass average size of the final DNA probe product synthesized as well as the overall rate of the nick translation reaction. Decreases in the amount of exogenous DNase I used in nick translation reactions produced significant decreases in the amount of hairpin loop structures formed. Hairpins could be effectively removed from nick-translated DNAs by employing hydroxylapatite column chromatography. Strategies to reduce hairpin formation during nick translation and the removal of hairpins from nick-translated DNAs are presented.