The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation.

The versatility of insertional inactivation of beta-galactosidase activity for subcloning and sequencing has been enhanced by combining a chemically synthesized oligonucleotide which specifies nine 6-bp-cutter restriction sites including BglII, XhoI, NruI, ClaI, SacI and EcoRV in various configurations with existing polylinkers to create a set of highly versatile cloning sites. These improved polylinkers have been inserted into plasmids (the pICs) for routine cloning of double-stranded DNA, and into chimeric phage/plasmids (the pICEMs) for biological production of single stranded DNA. The most versatile polylinker specifies 17 restriction sites in the beta-galactosidase alpha-complementing gene fragment. One of the new polylinkers was inserted into M13 DNA to produce a vector (M13mIC7) with nine cloning sites.

Thermodynamic interactions in the glutamate dehydrogenase-NADPH-oxalylglycine complex.

The enzyme-reduced coenzyme-alpha-ketoglutarate ternary complex is a critical intermediate in the glutamate dehydrogenase-catalyzed reaction. Oxalylglycine, a structural analog of alpha-ketoglutarate which contains an amide carbonyl group in place of a reducible ketone group, is one of the few compounds known to complete with alpha-ketoglutarate itself. In order to examine the role of the ketone group of alpha-ketoglutarate in the ternary complex, we have carried out a calorimetric study of the corresponding oxalylglycine ternary complex, determining the complete delta H, delta G, delta S, and delta Cp profiles and the corresponding interaction parameters for that complex and have compared the various parameters with the corresponding ones previously reported for the alpha-ketoglutarate ternary complex. While the overall delta G values of the two ternary complexes differ only slightly, the enzyme-NADPH-oxalylglycine ternary complex appears to achieve much of its stability from a very tight enzyme-oxalylglycine binary complex with little or no contribution from favorable interactions in the ternary complex, while the alpha-ketoglutarate ternary complex appears to achieve the same stability by a large interaction starting from a very weak enzyme-alpha-ketoglutarate binary complex. Consideration of the enthalpic profiles, however, show that this delta G-derived picture is deceptive. The excess binding energy which stabilizes the oxalylglycine binary is in fact due to hydrogen bonding of the amide group of oxalylglycine to the enzyme; in forming the ternary complex, this hydrogen bonding is lost in favor of forming an oxalylglycine-NADPH interaction, which is very similar to the alpha-ketoglutarate-NADPH interaction which stabilizes the alpha-ketoglutarate ternary complex. We conclude that the alpha-ketoglutarate-NADPH interaction must depend on either hydrogen bonding to or steric hindrance by the ketone group and that the existence of this energetically large interaction cannot be ascribed to imine formation between the keto group and enzyme. These findings also indicate the locus on the reaction coordinate where the reduced coenzyme plays a critical role, a role other than its obvious function as a hydride donor.