Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography.
We describe methods for the complete analysis of cellular nucleotides from as few as 10(6) 32Pi-labeled cells in a simple 2-day experiment. Nucleotides are extracted with acid, neutralized, and resolved by two-dimensional thin layer chromatography on polyethyleneimine cellulose. In the first dimension the nucleotides are separated based on the negative charge of their phosphate groups (i.e. cyclic, mono-, di, and triphosphates) and in the second dimension on their content of nucleobases (i.e. Ura, Cyt, Thy, Gua, and Ade). Because the separation is logical, one can predict the chromatographic migration of most nucleotides. By running standards we have determined the chromatographic location of over 90 biologically important nucleotides, nucleotide derivatives, and modified nucleotides from tRNA. We also developed a set of enzymatic and chemical methods to be used in conjunction with the chromatographic separations for verifying the identity of nucleotides and characterizing novel nucleotides. In this paper we use these methods to analyze and inventory the nucleotide content of Salmonella typhimurium in balanced log phase growth. Other potential uses of the method are also described.
D015720:5'-Nucleotidase; D000469:Alkaline Phosphatase; D002482:Cellulose / Q000031:analogs & derivatives; D002855:Chromatography, Thin Layer / Q000379:methods*; D004720:Endonucleases; D004722:Endoribonucleases; D006863:Hydrogen-Ion Concentration; D009708:Nucleotidases; D009711:Nucleotides / Q000302:isolation & purification*; D010727:Phosphoric Diester Hydrolases; D010761:Phosphorus Radioisotopes; D011094:Polyethyleneimine / Q000031:analogs & derivatives; D012260:Ribonucleases; D012486:Salmonella typhimurium / Q000032:analysis*; D015719:Single-Strand Specific DNA and RNA Endonucleases
D009711:Nucleotides; D010761:Phosphorus Radioisotopes; C018367:polyethyleneimine-cellulose; D011094:Polyethyleneimine; D002482:Cellulose; D004720:Endonucleases; D004722:Endoribonucleases; D012260:Ribonucleases; C022543:ribonuclease T(2); D009708:Nucleotidases; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D010727:Phosphoric Diester Hydrolases