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Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7°C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.

Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7 °C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.

Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue–purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4–7°C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.

ABSTRACTThe micronuclear DNA of Paramecium tetraureliais estimated to contain over 50,000 short DNA elements that are precisely removed during the formation of the transcriptionally active macronucleus. Each internal eliminated sequence (IES) is bounded by 5'-TA-3' dinucleotide repeats, a feature common to some classes of DNA transposons. We have developed an in vivo assay to analyze these highly efficient and precise DNA excision events. The microinjection of a cloned IES into mating cells results in accurately spliced products, and the transformed cells maintain the injected DNA as extrachromosomal molecules. A series of deletions flanking one side of a 28-bp IES were constructed and analyzed with the in vivo assay. Whereas 72 bp of DNA flanking the eliminated region is sufficient for excision, lengths of 31 and 18 bp result in reduced excision and removal of all wild-type sequences adjacent to the TA results in complete failure of excision. In contrast, nucleotide mutations within the middle of the 28-bp IES do not prevent excision. The results are consistent with a functional role for perfect inverted repeats flanking the IES.

The micronuclear DNA of Paramecium tetraureliais estimated to contain over 50,000 short DNA elements that are precisely removed during the formation of the transcriptionally active macronucleus. Each internal eliminated sequence (IES) is bounded by 5′-TA-3′ dinucleotide repeats, a feature common to some classes of DNA transposons. We have developed an in vivo assay to analyze these highly efficient and precise DNA excision events. The microinjection of a cloned IES into mating cells results in accurately spliced products, and the transformed cells maintain the injected DNA as extrachromosomal molecules. A series of deletions flanking one side of a 28-bp IES were constructed and analyzed with the in vivo assay. Whereas 72 bp of DNA flanking the eliminated region is sufficient for excision, lengths of 31 and 18 bp result in reduced excision and removal of all wild-type sequences adjacent to the TA results in complete failure of excision. In contrast, nucleotide mutations within the middle of the 28-bp IES do not prevent excision. The results are consistent with a functional role for perfect inverted repeats flanking the IES.

The germline chromosomes in Paramecium and other ciliated protozoa contain regions of DNA that are excised and eliminated during the development of a new macronuclear genome. Paramecium tetraurelia internal eliminated sequences (IESs) are invariably flanked by a 5′-TA-3′ dinucleotide sequence that is part of a larger 8-bp terminal inverted-repeat consensus sequence. Both features, the absolutely conserved 5′-TA-3′ and the remaining 6-bp terminal inverted repeat, are shared with the mariner/Tc1 class of transposons. In this article we describe a mutant cell line (AIM-2) defective in excision of a single IES from the coding region of the A51 surface antigen gene. Excision of the 370-bp IES6649 is prevented by a single A to G transition in the invariably conserved 5′-TA-3′ dinucleotide. Failure to excise IES6649 also revealed a 29-bp IES located inside IES6649. Additional experiments with the previously isolated AIM-1 mutant, which also contains an internal IES, shows that alternate excision using the wild-type end of IES2591 with an end from the internal IES is extremely rare or nonexistent. These results indicate that IESs are discrete elements whose excision depends upon nucleotides located within the consensus sequence, but also suggest that additional information is required to match one end of an IES with its excision partner.