Separation and some properties of distinct classes of newly-formed ribonucleic acid from animal cells

678

BIOCHIMICA ET BIOPHYSICA ACTA

Preliminory Notes PN 91033 Seporation ond some properties of distinct closses of newly-formed ribonucleic ocid from onimol cells As shown previously, newly-formed (rapidly-labeled) R N A of animal cells consists of D - R N A (-RNA with DNA-like base composition, messenger RNA) and R - R N A (RNA with ribosomal RNA-like base composition), which could be separated by thermal phenol fractionationl, 2. The heavy (45-35 S) radioactive material was predominantly observed in the R - R N A fraction 3. It contained two components scarcely resolved in sucrose gradient. Through modification of the previouslydescribed procedure it became possible to separate these components preparatively, as shown in Figs. I, 2 (a, b). At 35-4 o° (e.g. in temperature intervals lO-35 ° or IO-45 °) only one labeled peak with s20 equal to 35-4 ° S was liberated. In the 55 ° fraction (interval 45-55 °) another heavy component with s20 about 45 S was found. Both components were shown to be R-RNA, and they have been designated as R-RNA1 (45 S) and IR-RNA~ (40-35 S). Newly-formed D - R N A was recovered in the 55-63 ° fraction (Figs. I, 2c) and partly in the 45-55 ° fraction. The sedimentation properties of labeled D - R N A were similar in both of them. Newly-formed D - R N A consisted of heterogenous material (16-5o S) with a m a x i m u m in the 20-30 S zone. The ultraviolet absorption and radioactivity profiles did not coincide. Thus some metabolic heterogeneity of chromosomal D - R N A was observed. To elucidate the nature and the fate of different rapidly-labeled RNA's actinomyein chase experimentsd, a were performed (Figs. IC,). During 3 h after actinomycin blockage in vivo there was no transfer of radioactivity from one RNA fraction to another in Ehrlich ascites carcinoma cells. A considerable part of the newly-formed R - R N A (about 5 ° %) was degraded. D - R N A was relatively stable and remained unchanged during "chase". Although only part of the R - R N A was degraded, heavy R-RNA components disappeared completely (Figs. 3a, b, 4, 5b). In the 350(4 °0 ) fraction this process was followed by the appearance (or increase) of only one of the ribosomal RNA components, namely 28-S ribosomal RNA. On the other hand in the 55 ° fraction only I8-S ribosomal RNA appeared. Observation of the latter was complicated by the presence of labeled D-RNA in the 55 ° fraction. The following indicates the presence of I8-S IR-RNA: (I) coincidence of one of the radioactive components with the I8-S peak of ultraviolet absorption profile; (2) the presence of the D - R N A m a x i m u m in the heavier zone (it becomes clear b y comparing the radioactivity profiles of the 55 ° fraction, and of the pure D-RNA of the 63 ° fraction) (Figs. 3, 5b, c); (3) the intermediate base composition of labeled RNA of the 55 ° fraction after chase. These facts can be explained by suggesting the existence of two different polycystronic precursors: R - R N A 1 for I8-S ribosomal RNA, and R-RNA~ for 28-S ribosomal IRNA. However, results obtained in chase experiments are not conclusive, Biochim. Biophys. Acta, 91 (1964) 678-680

PRELIMINARY

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Biochim. Biophys. Acta, 91 ( 1 9 6 4 ) 6 7 8 - 6 8 0

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PRELIMINARY NOTES

and further experiments based on hybridization are needed to clarify the question about the number of ribosomal RNA precursors.

Institute o/ Radiation and Physical-Chemical Biology, Academy o/ Sciences o/the U.S.S.R.: Moscow (U.S.S.R.) Institute o/ Biological and Medical Chemistry, Academy o/ Medical Sciences o/the U.S.S.R., MoscoTe~ (U.S.S.R.)

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1 G. P. GEORGIEV AND V. L. ,'~,IANTIEVA, Biokhirniya, 27 (1962) 949. 2 M. I. LERMAN, V. L. MANTIEVA AND G. P. GEORGIEV, Dokl. Akad. Nauk. S S S R , 152 (1963) 744. G. P. GEORGIEV, O. P. SAMARINA, M. ]. LERMAN AND ]~I. N. SMIRNOV, Nature, 200 (1963) 1291. 4 R. P. PERRY, Proc. Natl. Acad. Sci. U.S., 48 (1962) 2179. 5 K. SCHERRER, H. LATHAM AND J. E. DARNELL, Proc. Natl. Acad. Sci. U.S., 49 (1963) 240.

Received July 27th, 1964 Biochim. Biophys. Acta, 91 (1964) 678-68o

PN 91038

Messenger ribonucleic acid activity in rabbit reticulocyte ribosomes When an erythroid cell reaches the reticulocyte stage it has lost its ability to synthesize RNA 1-4, but retains its ability to synthesize hemoglobin 5. This indicates that the genetic messenger is stable. Because of this stability and because reticulocytes synthesize predominantly hemoglobin 5, they should provide a good material for the isolation of a specific messenger RNA. Several investigatorsa,4, 6 have shown that the information for hemoglobin synthesis is located in the ribosomes of reticulocytes. It has further been reported that reticulocyte ribosomal RNA enhances amino acid incorporation into trichloroacetic acid insoluble material when added to either a reticulocyte cell-free system 7-12 or to an Escherichia coli cell-free system1°, ~3. In more specific experiments, ARNSTEIN AND COX8 have shown that this enhancing activity is associated with 29.8-S ribosomal RNA. Their RNA was prepared by a guanidine hydrochloride extraction procedure. MATHIAS et al. ~1, using the lithium chloride method for RNA isolation la, found that the I6-S, 28-S, and 4I-S components gave roughly equal enhancement when added to the reticulocyte cell-free system. SHAEFFER et al. ~3 and HARDESTY et al. 1° reported that isolation of reticulocyte ribosomal RNA by the phenol method failed to result in a purification of the messenger activity when Iractionated by sucrose density gradient centrifugation. This communication reports preliminary work on the isolation of a messenger RNA fraction from reticulocytes. Reticulocyte ribosomes were prepared by the method of LINGRELAND BORSOOK 15, modified only in that the reticulocyte lysing solution contained 3 mg/ml of bentonite. Ribonucleic acid was then prepared from these ribosomes by each of three methods, i.e., by phenol extraction similar to the technique of •IRENBERG AND MATTHAE118, by the lithium chloride extraction method of BARLOW et al. ~4, and by the guanidine hydrochloride method of Cox AND ,A_RNSTEIN17. Biochim. Biophys. Acta, 91 (1964) 68o 683