Gene expression of ascorbic acid-related enzymes in tobacco

GDP-D-mannose pyrophosphorylase (GMPase) and L-galactono-1, 4-lactone dehydrogenase (GalLDH) are key enzymes in L-ascorbic acid (AsA) biosynthesis of plants, and a full-length cDNA for GMPase was isolated from tobacco using PCR. Additionally, expression of GMPase, GalLDH and other AsA-related enzymes was examined in tobacco tissues and cultured BY-2 cells, and the relationship between their expression patterns and AsA content is discussed. It was found that the expression of GalLDH and GMPase mRNAs was markedly suppressed by loading AsA, suggesting that AsA concentration in the cells may regulate AsA biosynthesis. Moreover, the expression of GMPase and GalLDH mRNAs in tobacco leaf also suggested that AsA biosynthesis may be induced by light.     

Gene cloning and polymerase chain reaction with proliferating cell nuclear antigen from Thermococcus kodakaraensis KOD1

The gene encoding the proliferating cell nuclear antigen (PCNA), a sliding clamp of DNA polymerases, was cloned from an euryarchaeote, Thermococcus kodakaraensis KOD1. The PCNA homologue, designated Tk-PCNA, contained 249 amino acid residues with a calculated molecular mass of 28,200 Da and was 84.3% identical to that from Pyrococcus furiosus. Tk-PCNA was overexpressed in Escherichia coli and purified. This protein stimulated the primer extension abilities of the DNA polymerase from T. kodakaraensis KOD1 'KOD DNA polymerase'. The stimulatory effect of Tk-PCNA was observed when a circular DNA template was used and was equally effective on both circular and linear DNA. The Tk-PCNA improved the sensitivity of PCR without adverse effects on fidelity with the KOD DNA polymerase. This is the first report in which a replication-related factor worked on PCR.     

Mechanism of the Increase in Catalase Activity through Microbody Development in Wounded Sweet Potato Root Tissue

An increase in catalase activity accompanied by microbody developmentin wounded sweet potato root tissue was investigated with aspecific antibody against sweet potato catalase. The increasewas completely inhibited by cycloheximide. Analysis with singleradial immunodiffusion method showed that protein immunoprecipitatedby the antibody increased in wounded tissue, indicating theinvolvement of de novo synthesis of catalase protein in theactivity-increase. The activity-increase was, however, moreremarkable than the increase in immunoreactive protein and thisresults in an increase in specific catalase activity in woundedtissue, indicating the presence in intact tissue of an inertor less active protein, immunologically analogous to catalase.Actually, immunological analysis showed the presence in intacttissue of an immunoreactive protein which differed from activecatalase protein in the mobility on a polyacrylamide gel andprobably also in the molecular weight of subunit. The immunoreactiveprotein seemed to exist in a significant amount outside themicrobodies in intact tissue cells. Thus, there is a possibilitythat the increase in catalase activity in wounded tissue ispartly due to activation of the immunoreactive protein. (Received October 16, 1982; Accepted February 24, 1983)