A simple method for cloning genes involved in glucan biosynthesis: isolation of structural and regulatory genes for glycogen synthesis in Escherichia coli.
A simple and widely applicable method for cloning genes involved in glucan biosynthesis is described. An Escherichia coli genomic library was prepared in the low-copy plasmid, pLG339, and E. coli transformants from this library were screened by staining with iodine vapor. Colonies that stained darker than the control were isolated and characterized. The three classes of clones that were identified included: (i) plasmids encoding E. coli glycogen biosynthetic (glg) structural genes, (ii) clones that resulted in elevated glycogen levels, but did not encode glg structural genes or enhance the level of the first enzyme of the pathway, ADPglucose pyrophosphorylase (AGPP), and (iii) clones that enhanced the level of AGPP, but did not encode this enzyme. Two clones from the latter class also enhanced glgC'-'lacZ-encoded beta-galactosidase activity, and may encode factors that regulate the expression of glg structural genes. It should be possible to readily clone glycogen biosynthetic genes from other bacterial species via this method. The method could be made specific for a desired glg gene by using a recipient strain that is defective in the gene of interest.
Romeo
T
T
Department of Microbiology and Immunology, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth 76107.
Moore
J
J
Smith
J
J
D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D005809:Genes, Regulator / Q000235:genetics; D005816:Genetic Complementation Test; D005936:Glucans / Q000096:biosynthesis / Q000235:genetics*; D051599:Glucose-1-Phosphate Adenylyltransferase; D006003:Glycogen / Q000096:biosynthesis / Q000235:genetics*; D006006:Glycogen Synthase / Q000235:genetics; D007455:Iodine / Q000494:pharmacology; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D015183:Restriction Mapping; D001616:beta-Galactosidase / Q000235:genetics
D005936:Glucans; D011993:Recombinant Fusion Proteins; D006003:Glycogen; D007455:Iodine; D006006:Glycogen Synthase; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D001616:beta-Galactosidase