The sequence motif commonly called a Nudix box, represented by (GX 5 EX 7 REVXEEXGU) is the marker of a widely distributed family of enzymes that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we describe the cloning and characterization of an Arabidopsis thaliana cDNA encoding a Nudix hydrolase that degrades NADH. The deduced amino acid sequence of AtNUDT1 contains 147 amino acids. The recombinant AtNUDT1 was expressed in Escherichia coli and purified. In the presence of Mn 2؉ and the optimal pH of 7. 0, the recombinant AtNUDT1 catalyzed the hydrolysis of NADH with a K m value of 0. 36 mM. A V max of 12. 7 units mg ؊1 for NADH was determined. The recombinant AtNUDT1 migrated as a dimer on a gel filtration column. Biochemical analysis of recombinant AtNUDT1 indicated that the first characterized member of the Nudix family from A. thaliana is a NADH pyrophosphatase.
L i s t of abbreviations: GUS, b-glucuronidase; MS m e d i u m , Murashige and Skoog medium; PAT, phosphinothricin acetyl transferase; PPT, phosphinotricin.
AbstractAn optimized procedure for transformation of wheat with the use of a Biolistic Particle Delivery System PD S 1000/He to deliver ~breign DNA is described in detail. The bacterial uidA and bar genes (both driven by plant promoters) were utilized as the reporter and selectable marker genes, respectively. Moderately high gas pressure appeared to be most important to achieve the highest level of transient GUS expression in target tissues. There was, however, no apparent correlation between transient and stable GUS expression. The presence of telomeric DNA sequences in an uidA gene-containing vector did not influence transient GUS expression but, apparently, prevented its stable expression. Mechanical lesions caused by the bombardment (tungsten particles) seemed to be less severe when embryo-derived calli, instead of freshly excised immature embryos, were used as the target tissue. The limited ability of callus cells for regeneration, together with a restricted number of cells that receive the foreign DNA by particle bombardment, result in a low efficiency of wheat stable transformation.
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