title: Cloning of the ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) structural genes from Salmonella typhimurium LT2.
abstract: The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D051599:Glucose-1-Phosphate Adenylyltransferase; D006006:Glycogen Synthase / Q000235:genetics* / Q000276:immunology; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000276:immunology; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D006006:Glycogen Synthase; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D004262:DNA Restriction Enzymes