Studies on the Isolation of Messenger RNA

Citation

Lyon, Alexander Newell (1969) Studies on the Isolation of Messenger RNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/HPJ4-J340. https://resolver.caltech.edu/CaltechTHESIS:06072017-100016945

Abstract

The rapidly-labeled RNA fraction of E. coli has been purified approximately 15-fold on benzoylated DEAE cellulose columns (BC). It is metabolically unstable (as shown by a pulse/chase experiment) and is considered to represent mRNA. The yield of pulse-labeled RNA is about 70% and comprises 4-5% of the RNA of the cell.

The true size distribution of this RNA, determined by sedimentation in a denaturing solvent (99% DMSO), does not change during purification. This result indicates that neither degradation nor selection for molecules of a particular size has occurred. Upon sedimentation of the final preparation in DMSO, the distribution of pulse label is the same as that of RNA mass, indicating nearly complete separation from longer-lived RNA components.

The isolation of globin-specific mRNA from rabbit reticulocytes has been attempted, both by a modification of BC chromatography and by the previously published sucrose gradient method of Marbaix, Burny, and Chantrenne, but the results (in both cases) were inconclusive. The final preparation sedimented heterogeneously, with an estimated mean sedimentation coefficient (s₂₀,w) of 8.4 S. Tests of the possible identity of this material as mRNA by biological assay in a cell-free protein synthesizing system have not been attempted.

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