Polysomes from thyroids incubated in vitro

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421

zymic attack on the ribosomal nucleic acid. This would account for the large RNA losses observed during starvation of this organism. Several strains of N. crassa including the one used in this study have been found to accumulate polyphosphate when deprived of an essential amino acid 1. Chelation of cellular magnesium by polyphosphate may be contributing to the lability of the ribosomes in vivo. We thank Dr. E. S. FIALA for some of the protein analyses. This work was supported by a grant from the U. S. Public Health Service (GM-o9999). Department o~ Physiology and Biochemistry and the Bureau o/ Biological Research, Rutgers - the State University, New Brunswick, N. J. (U.S.A.)

ETHEL W. BERNARD

SOMBERG

J.

SALMON

F R A N K F . DAVIS

I F. M. HAROLD, Biochim. Biophys. Acta, 45 (196o) 172. 2 E. W . SOMBERG, AND F. F. DAVIS, Biochim. Biophys. Acta, lO8 (i965) 137. 3 N. H. HOROWITZ, J. Biol. Chem., 171 (1947) 255. 4 W. C. SCHNEIDER, J. Biol. Chem., 161 (1945) 293. 5 A. H. BROWN, Arch. Biochem. Biophys., i i (1946 ) 269. 6 A. G. GORNALL, C. J. BARDAWELL AND M. M. DAVID, J. Biol. Chem., 177 (1949) 751. 7 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 8 E. REICH AND S. TSUDA, Biochim. Biophys. Acta, 53 (1961) 574. 9 R. STORCK, Biophys. J . , 3 (1963) i.

Received February 9th, 1966 Revised manuscript received July 8th, 1966 Biochim. Biophys. Acta, 129 (1966) 419-421

BBA 93165

Polysomes from thyroids incubated in vitro Most studies on the effects of hormones on protein and nucleic acid synthesis in target tissues have been performed in vivol, ~. Such studies are technically easy and may be kept within the limits of normal physiology. However, the complexity of whole organisms makes it questionable to assume that observed effects of hormones or drugs are due to a direct action of these compounds on the investigated tissues. An ideal system would therefore be an in vitro preparation which would retain the main advantages of the in vivo system, namely stability and differentiation. Sheep thyroid slices incubated in vitro may constitute such a system; indeed these slices remain well differentiated and metabolically stable for 24 h in vitroD,4. The purpose of this work was to investigate the possibility of studying polysomes in such an in vitro preparation. The preparation of sheep thyroid slices and the incubation procedures have been previously described s. An important modification of the technique was the immediate immersion of thyroids collected from dying animals in the complete inBiochim, Biophys. Acta, I29 (1966) 4 2 1 - 4 2 4

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cubation medium at 2 °. After the incubation, the slices (approx. 2 g per analysis), were chopped into cubes 0.26 mm thick with the McIlwain tissue chopper (MIcKLE, GROMSHALL, Survey (U.K.)). The resulting mash was added to 9 ml of a solution containing 25 ° mM sucrose, 5° mM Tris-HC1 buffer (pH 7.4), 25 mM KC1 and 5 mM MgC12; it was then homogenized by one stroke of the loose-fitting teflon pestle of a Potter-Elvehjem homogenizer. Ribosomes were prepared from the homogenate by the technique of WETTSTEIN, STAEHELIN.ANDNOLLe. Except for the incubation, all the manipulations were carried out in the cold room at o °. A kinetic study of the centrifugation of monosomes and polysomes at lO5 ooo ×g in double-layered sucrose showed that a 2-h centrifugation was sufficient to sediment virtually all the polysomes and 7° % of the monosomes. The duration of this centrifugation was therefore shortened from 4 to 2 h. After the density-gradient centrifugation, the absorbances of the various layers in the tube were measured at 260 m/~ in a continuous-flow cell in a Zeiss PMQII speetrophotometer. Fractions of 1o drops were collected at the outlet of this cell in scintillation counter vials containing I ml distilled water. After addition of 15 ml BRAY~ solution, the vials were counted for radioactivity in a/~ scintillation counter Packard Model 3003. Shifts in the ribosome patterns were evaluated by changes in the ratios P / ( P + M ) where P and M were the absorbances corresponding to polysomes and monosomes, respectively (Fig. I). This ratio provides a sensitive index of the integrity of the protein-synthesizing machinery s. Incorporations of labeled precursors in polysomes were expressed as counts/rain per cm ~ of the polysomal peak area. Total incorporation of labeled precursors in proteins and nucleic acids of incubated slices was measured by a method derived from MANS AND NOVELLI~,9. [aH]orotic acid, Jail]uracil and [aH]uridine were purchased from the Radiochemical Centre (Amersham, England), [aH]cytidine and 14C uniformly labeled amino acid mixture from N.E.N. {Boston, Mass., U.S.A.) and calf serum and medium I99 from Burroughs Wellcome {London, England). Actinomycin D was generously furnished by Merck, Sharp and Dohme (Rahway, N.J., U.S.A.). Slices from the same animals were incubated for 2 h in 4 different incubation media. The P / ( P + M ) ratios were o.43, o.88, o.48, and 0.74 for slices incubated in Krebs-Ringer-phosphate, Krebs-Ringer-bicarbonate and Krebs-Ringer-Tris buffer 5 and in the Campagne-Gruber medium x°, respectively. The addition of culture medium 199 (ref. II), or of Io % calf serum to the Krebs-Ringer-bicarbonate buffer increased the P / ( P + M ) ratios from o.58 to o.62 and o.65, respectively. Krebs-Ringer-bicarbonate buffer containing IO o~ ,o calf serum, and culture medium 199 was therefore used for the following experiments. Slices from the same thyroids incubated similarly in 3 different flasks had P/(P +M) ratios of o.38, o.4 ° and o.4 I. Incubation of thyroid slices did not markedly change the ribosome pattern in these slices. P / ( P + M ) ratios before and after a 4-h incubation were o.81 and o.69 for one thyroid and o.68 and o.66 for another, respectively. Thyroid slices incorporated ~4C-labeled amino acids in the ribosomes, mainly at the polysomal level (Fig. I). This is consistent with the current theory that these aggregates are the functional units in protein synthesis ~. It was also at the polysomal level that [aH]cytidine and [aH]uridine, but not [~4C]orotate or jail]uracil were significantly incorporated in ribosomes. This incorporation was completely inhibited Biochil~. l?iophys. Acla, ~29 (1960)421

424

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by actinomycin (5 yg/ml); it was therefore due to a new synthesis of RNA and not to the turnover of the terminal portion of tRNA. When polysomes labeled with [3H!uridine were partially digested with ribonuclease (o.oi yg/ml, 20 rain, 37°), most of the radioactivity was found in the soluble fraction, and not in the monosomes (Fig. I). These facts support tile hypothesis that IaH]uridine was incorporated into the m R N A which binds the ribosomes in polysomes. The pattern of incorporation of precursors in the ribosomes of thyroid slices in vitro was therefore similar to the pattern observed in rat liver after an injection of these compounds in vivo 13.

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Fig. I. R i b o s o m e p a t t e r n of sheep t h y r o i d slices i n c u b a t e d for 3 h in t h e p r e s e n c e of [3H~uridine (3.8/~C/ml). A 14C-labeled a m i n o acid m i x t u r e (0.38/~C/ml) w a s a d d e d to t h e i n c u b a t i o n m e d i u m 3 ° m i n before t h e en d of t h e i n c u b a t i o n . Sucrose a b s o r p t i o n a t 260 m # a c c o u n t s for t h e slope of t h e abscissae. A, N o r m a l r i b o s o m e p a t t e r n . The r a t i o P / ( P + M ) c o r r e s p o n d s to t h e r a t i o of t h e c l e a r a r e a to t h e t o t a l a r e a of t h e r i b o s o m e s . B, R i b o s o m e p a t t e r n of t h e s a m e r i b o s o m e s a f t e r a 2o-min i n c u b a t i o n a t 37 ° in t h e p r e s e n c e of o . o i / ~ g / m l of r i b o n u c l e a s e . - - , Absorbance at 260 m # ; × - × , c o u n t s / r a i n 3H ~ [3H]uridine i n c o r p o r a t i o n in t h e r i b o s o m e s ; @ - @ , c o u n t s / m i n 14C ~ 14C-labeled a m i n o acid i n c o r p o r a t i o n in t h e r i b o s o m e s .

The rate of incorporation of [aHltyrosine into the proteins of thyroid slices was constant during an 8-h incubation. [aHlcytidine incorporation into total RNA was also perfectly stable from the 2nd to the 8th h of incubation. There was a burst of increased incorporation during the first 2 h of incubation, however. Values for the incorporation of [3H]uridine at the polysomal level during the 2 h-4 h 30 min and the 4 h 45 min-7 h 15 rain periods were 276 and 230 counts/min, respectively. The stability of protein and RNA synthesis is therefore satisfactory in incubated thyroid slices. In conclusion, the methods described in this article permit the study of polysomes in thyroid slices incubated in vitro with results comparable with those of studies in vivo. This work was carried out under contract Euratom, University of Pisa, University of Brussels, No. 026 63-4 BIAC. The authors would like to thank Prof. H. CHANTRENNE for his advice in these studies and Miss CH. BORRE¥ for the preparation of the manuscript. Laboratoire Central de Mddecine Nucldaire, Universild Libre de Bruxelles, Brussels (Belgium)

R. E. LECOCQ J. E. DUMONT

Biochim. Biophys. Acta, 129 (1966) 421-424

424

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I G. LITVACK AND D. KRITCHEVSKY, Actions o] Hormones on Molecular Processes, J. \Viley, New York, 1964 . 2 Symposium on hormonal control o[ protein biosynthesis, J. Cell. Comp. Physiol., 66 (1965) Suppl. i. 3 R. W'. SEED AND I. H. GOLDBERG, Proc. Natl. Acad. Sci. U.S., 5o (1963) 275. 4 J. NUNEZ, J. MAUCHAMP, V. MACCHIA AND J. ROCHE, Biochim. Biophys. Acta, lO 7 (1965) 247. 5 J- E. I)UMONT, Ann. Soc. Roy. Sci. Med. Nat. Bruxelles, 18 (1965) lO6. 6 F. O. WETTSTEIN, T. STAEHELIN AND H. ~N~-OLL,Nature, 197 (1963) 43 o. 7 G. A. BRAY, Anal. Biochem., i (196o) 279. 8 T. E. WEBB, G. BLOBEL AND R. VAN POTTER, Cancer Res., 24 (1964) 1229. 9 R. J. MANS AND G. D. NOVELLI, Arch. Biochem. Biophys., 94 (1961) 48. io R. N. CAMPAGNE AND M. GRUBER, Biochim. Biophys. Acta, 55 (1962) 353. I I J. F. MORGAN, H. J. MORTON AND R. C. PARKER, Proc. Soc. Exptl. Biol., 73 (195 °) I. 12 A. RICH, J. R. WARNER AND H. M. GOODMAN, Cold Spring Harbor Syrup. ~)uant. Biol., 28 (1963) 269. 13 R. R. HOWELL, J. N. LOEB AND G. M. TOMKINS, Proc. Natl. Acad. Sci. U.S., 52 (1964) 1241.

Received June 8th, 1966 Biochim. Biophys. Acta, 129 (1966) 421-424

BBA 93161

Electrophoretic separotion of the mononucleotides from on olkaline hydrolysoteof R N A The classical procedure for determining the nucleotide composition of RNA (refs. 1, 2), namely, by alkali digestion, neutralization with perchloric acid, and separation of the nucleotides has undergone very few changes a-5. The present report describes a buffer system for high-voltage paper electrophoresis that permits the separation of mononucleotides without prior neutralization of the alkali used to hydrolyze the RNA. To separate the nucleotides, sodium acetate at different ionic strengths and pH values were used for the electrophoresis. At pH 3.8 (0.4 M), the nucleotides migrated from the origin and the resolution was good (Fig. IA). It was important, however, to adjust the pH of the system very carefully. At values below 3.7 (Fig. IA), CMP did not migrate from the origin (the analysis would be highly inaccurate if the RNA samples contained any labeled macromolecules, see Fig. IB), while at higher values it became increasingly difficult to separate GMP and UMP. The molarity of the buffer appeared to have little effect on the resolution of the nucleotides, although o.2 M buffer (Fig. IB) was preferred to 0.4 M since the amperage was lower, thereby decreasing the heating of the paper during the electrophoresis. Since the RNA preparations that were used in this study contained 5-Io % DNA migrating in the I6-2o-S region of sucrose gradients °, the effect of this contamination on the base analysis was determined. Fig. IB shows the separation of the nucleotides after deoxyribonuclease treatment. The base composition of these two samples was similar to that reported for this RNA (ref. 7). In studies on RNA from potato tubers or pea stems, it was observed that CMP would not migrate from the origin as a discrete spot, resulting in a polymer A contamination of CMP. This problem was eliminated by neutralizing the alkali with either HC1 or perehloric acid. Use of HC1 to neutralize the alkali resulted in the formation Biochim. Biophys. Acta, 129 (1966) 424-426