Abstract:

UDP-Glc: protein transglucosylase (UPTG), is an autocatalytic protein which undergoes self-glucosylation in an UDP-glucose and Mn2' dependent reaction. UPTG can initiate in vitro protein-bound a-glucan synthesis and was postulated as the protein that prunes starch biosynthesis in vivo. Specific antibodies raised against UPTG purified from potato (Solatium tuberosum L.) tubers were used to clone a cDNA from a swelling stolon tip potato library. RNA hybridization studies and western blot analysis indicates that UPTG mRNA is expressed in all potato tissues analized as expected for a protein involved in starch synthesis. The cloned sequence was expressed in E. coli. The expressed polypeptide has an apparent Mr of about 40.000, close to that reported for UPTG and was recognized in a western blot by the specific antibodies against purified potato UPTG. The recombinant UPTG was labeled after incubation with UDP-['4C]ghicose and Mn2 jinHi-ating that it was enzymalically active. Furthermore, the recombhunt enzyme showed specificity for UDPglucose as the glucosyl donor, activation by Mn2 and ability to transfer xylose from UDPxylose, as previously reported for the UPTG purified from potato tuber. The availability of the UPTG cDNA will allow interesting experiments to be performed. Physiological analysis of UPTG transgenic plants should allow the investigation of the role of UPTG in starch synthesis in greater detail. This woik was supported by an International Cooperation Agreement between CONICET (Argentine) and FNRS (Belgium), Universioad de Buenos Aires (Ex-305) and by UNESCO (Short Term Fellowship in Biotechnology to S.N.B.).