On the Extraction of Ribonucleic Acid with Template Activity from Barley Embryos Using Diethyl Pyrocarbonate as Nuclease Inhibitor

Using diethyl pyrocarbonate as enzyme inhibitor, RNA was extracted from embryos of germinating barley kernels. The template activity of this RNA was tested by its capacity to stimulate 14Cphenylalanine incorporation into trichloroacetic acid insoluble protein synthesized in an Escherichia coli cell free system. The template activity was compared with that of RNA isolated from barley embryos by other methods of extraction. The RNA extracted by the diethyl pyrocarbonate method, omitting sodium dodecyl sulfate, was found to have better template activity than the others, and was equvalent or superior to tobacco mosaic virus RNA isolated by the phenol method. Sucrose density gradient sedimentation of the RNA yielded fractions with still higher specific template activity. Reextraction with phenol of the RNA extracted by the diethyl pyrocarbonate method reduced the template activity of the RNA. Native and denatured DNA were also found to decrease the template activity of RNA.RNA extracted from kernels germinated in the presence of 5-fluorodeoxyuridine had a reduced template activity indicating that this compound may inhibit mRNA synthesis, possibly indirectly by blocking strand separation in DNA synthesis.