Isolation of L-cell messenger RNA which lacks poly(adenylate)

Abstract

It has been found that centrifugation of (ethylenediamine)tetraacetic acid dissociated L-cell polyribosomes at 25 degrees C through preformed CS2SO4 density gradients containing 15% dimethyl sulfoxide resolves them into two distinct classes of particles that contain ribosomal and messenger RNA, respectively. Ribosomal RNA bands at a higher density than mRNA, presumably because it is more extensively stripped of protein by the action of CS2SO4 than mRNA. Undergraded RNA can be recovered from the gradients by trapping the particles on nitrocellulose filters and then eluting at 60 degrees C with a solution containing formamide and sodium dodecyl sulfate. When cells were labeled with [3H]adenosine, either in the presence or absence of a dose of actinomycin D which selectively inhibits rRNA synthesis, and their polyribosomes centrifuged through CS2SO4 density gradients, a large amount of the radioactivity recovered from the messenger-ribonucleoprotein region of the gradient had the size distribution of mRNA, but lacked poly(adenylate) (poly (A)). This material constituted 29-31% of the total mRNA. Histone mRNA was the most prominent component of poly(A)-lacking mRNA. However, 50-65% of the poly(A)-lacking mRNA was of larger size than histone mRNA.