The effect of polyamines on the enzymatic degradation of ribosomes

BIOCHIMICAET BIOPHYSICAACTA

43

BBA 95845

T H E E F F E C T OF P O L Y A M I N E S ON T H E ENZYMATIC D E G R A D A T I O N OF RIBOSOMES v. A. ERDMANN*, G. A. THOMAS'*, J. W. NORTON AND E. J. HERBST Department of Biochemistry, Spaulding Life Science Building, University o/ New Hampshire, Durham, N.H. (U.S.A.) (Received October 9th, 1967)

SUMMARY

I. Broken cell extracts of Escherichia coli and ribosomes isolated from such extracts were incubated in o.I M "Iris buffer, pH 7.5-8.1, or in buffer containing 0.05 M EDTA. A rapid enzymatic degradation of r R N A occurred and the acid-soluble products of these reactions were identified as nucleoside 3'-phosphates. 2. Spermine and spermidine at low concentrations ( o . I - i mM polyamine) inhibited the autodegradation of the ribosomes in o.I M Tris but did not inhibit ribosome degradation when the polyamine concentration was increased. When EDTA was wesent, the degradation of the ribosomes was completely inhibited b y I-IO mM polyamine. 3. The inhibition by polyamines of the latent ribonuclease (ribonuclease I) released by E D T A could not be demonstrated with purified rRNA and ribonuclease I derived from E. coli ribosomes. 4. Low concentrations of the polyamines appear to stabilize the ribosomes and prevent the release of ribonuclease I while high concentrations of the polyamines destabilize the ribosomes and release ribonuclease I.

INTRODUCTION The enzymatic degradation of R N A in Hemophilus parain/luenzae is inhibited by polyamines 1. A similar effect of polyamines on the degradation of RNA in broken cell preparations and ribosomes of Escherichia coli B was demonstrated by THOMAS2 but the mechanism by which the polyamines reduce the enzymatic degradation of R N A and the identity of the enzyme(s) was not established. The recent characterization of the ribonucleases of E. coli ~,4 clearly differentiates a ribonuclease I (EC 2.7.7.17) which cleaves all internucleotide bonds to nucleoside 3'-phosphates and a ribonuclease I I (EC 3.14.1, phosphodJesterase) which produces nucleoside 5'-phosphates and which is dependent upon divalent cations in contrast to the inhibition of ribonuclease I by the same cations. Interference with the enzymatic degradation * Present address: Max Planck Institut ffir experimentelle Medizin, G6ttingen, Germany. Present address: School of Medicine, The Johns Hopkins University, Baltimore, Md. U.S.A. Biochim. Biophys. Acta, 157 (1968) 43-5I

44

v . A . ERDMANN el a[.

of RNA by polyamines is not unexpected since there is considerable evidence of electrostatic interactions between these basic compounds and polynucleotides which affect physical-chemical and biological proporties of both RNA and DNA (ref. 5)The concentration of polyamines is an important factor in polyamine-nucleic acid interactions. In this investigation, both inhibition and stimulation of nuclease activity b y low and high concentrations respectively of the polyamines were observed. While these concentration-dependent effects of spermidine and spermine are readily demonstrable with cell preparations and ribosomes containing ribonuclease I, purified enzyme and ribosomal RNA (rRNA) are not similarly affected b y comparable amounts of the polycations. METHODS AND MATERIALS

Bacterial strains and culture methods Escherichia coli strain B was grown in glucose-salts medium 6 with shaking at 35 °. E. coli MRE6oo, a ribonuclease I - strain selected b y CAMMACK AND W A D E 7, was obtained from Dr. W. GILBERT,H a r v a r d University and was grown in the glucose-salts medium supplemented with 15 mM glutamate. The cells were harvested during exponential growth (I. lO 9 cells per ml) and washed twice with o.oi M Tris buffer (pH 7.5). Washed cell pellets were frozen in o.5-g lots and held at --20 ° until immediately prior to preparation of cell extracts. Preparation o/cell extracts and ribosomes Frozen cell pellets were ground with alumina (Norton Co., Worcester, Mass.) b y the method of TISSIf~RES et al. 8, and ribosomes were prepared from cell extracts in o.oi M Tris (pH 7.4) b y centrifugation at IO ooo x g for 2 h in a Spinco Model L-2 ultracentrifuge. Ribosomes were suspended in cold o.I M Tris buffer (pH 8.1) and utilized immediately in autodegradation studies, or ribosome pellets were frozen and held overnight at --20 °. Cell extracts for autodegradation studies were prepared in cold o.I M Tris buffer (pH 8.1) and used immediately or frozen and held overnight in tile same buffer at --20 ° . A utodegradation o/cell extracts and ribosomes The incubation system for cell extracts contained o.5 ml cell extract (approx. 3 absorbance units at 260 m/~) in a final volume of 3 ml o.i M "Iris buffer (pH 8.1). After incubation at 37 ° for 30 min the tubes were placed in an ice b a t h and precipitated with 0.5 ml of 0.75 % uranyl acetate in 24 % perchloric acid. The absorbance of the supernatant (acid-soluble fraction) was determined at 260 m#. Approx. I absorbance unit of ribosome suspension was incubated in a final volume of 1.2 ml of o.I M Tris buffer, (pH 8.1). After 30 min at 37 ° the tubes were placed in ice and precipitated with 0.2 ml of 0.75 % uranyl acetate in 24 % perchloric acid and the absorbance of the acid-soluble fraction was determined at 260 m/~. Ribonuclease I assay procedure Ribonuclease I was prepared from purified ribosomes s b y the procedure of SPAHR AND HOLLINGWORTHa. Ribosomal ribonucleic acid (rRNA) was prepared from Biochim. Biophys. Acta, I57 (1968) 43-51

POLYAMINES AND RIBOSOME DEGRADATION

45

o.5 g of purified ribosomes b y the phenol procedure of KURLAND9. The activity of ribonuclease I was determined in a reaction mixture which contained: I mg rRNA, o.oi mg ribonuclease I and o. I M Tris buffer (pH 8. I) in a total volume of 1.2 ml. After incubation at 37 ° for 30 rain the tubes were chilled, and precipitation with uranyl acetate-perchloric acid was carried out as previously described.

Identi[ication o/end products o/nuclease action Cell extracts or ribosome suspensions (approx. IOO absorbance units at 260 m#) were incubated in 5 ml of o.I M Tris buffer (pH 8.1) or in Tris buffer containing either o.oi M E D T A or 8 mM spermidine for 15o min at 37 °. The incubations were stopped b y adding cold trichloroacetic acid to 5 % final concentration, and, after centrifugation, the trichloroacetic acid was extracted with diethyl ether. The ether was removed with N 2 and the sample was neutrahzed. Nucleotides were separated b y the ion-exchange method of COHN1° and were identified b y comparison with elution diagrams from columns loaded with appropriate nucleotide standards. Confirmation of the identity of the products derived from the autodegradation of cell extracts and ribosomes was accomplished b y a modification of the chromatographic procedure of PLESNER11. Thin-layer plates were prepared with W h a t m a n cellulose CC4I powder and, after drying at room temperature, the plates were spotted with 50 #1 (approx. I absorbance unit at 260 m#) of the unknown nucleotide mixtures and appropriate nucleoside 3'-phosphate and nucleoside 5'-phosphate standards. The plates were developed with I M ammonium acetate containing 5 % sodium metaborate-96 ~o ethanol-conc. NH4OH (6:15:2, b y vol.). Nucleoside 3'-phosphates migrate with this solvent mixture while nucleoside 5'-phosphates and oligonucleotides remain at the origin. The presence of oligonucleotides was ascertained b y incubating an aliquot of the nucleotide mixtures from autodegradation experiments in 0. 3 M N a O H at 37 ° overnight. The N a O H was neutralized and the hydrolyzed samples were compared with unhydrolyzed nucleotide mixtures on the same thin-layer plates. RESULTS

EHect o[ polyamines on the degradation o[ RNA in cell extracts Ribonucleic acid is rapidly degraded in broken cell extracts of E. coli B during incubation in o.I M Tris buffer (pH 8.1) or in Tris buffer containing 0.05 M EDTA. These enzymatic degradation processes are inhibited b y the polyamines, spermine and spermidine. The data illustrating the effect of various concentrations of spermidine on the degradation of R N A in cell extracts appears in Table I. Spermidine is inhibitory at concentrations of o.I to I.O mM but a reactivation of the enzymatic activity occurs at higher concentrations. Cell extracts prepared in o. i M Tris buffer and frozen overnight prior to incubation at 37 ° are not protected, however, by any concentration of spermidine. The polyamine is an effective inhibitor of the breakdown of ribonucleic acid in cell extracts containing 0.05 M E D T A and the inhibition is complete at approx. 5-1o mM spermidlne with no reactivation of the enzyme system at higher concentrations. Experiments in which spermine was added to either flesh or frozen cell extracts, or to cell extracts containing EDTA, yielded very similar results (Table II). The Biochim. Biophys. Acta, 157 (1968) 43-51

46

v.A. ERDMANN e[ al.

TABLE

I

EFFECT OF SPERMIDINE ON DEGRADATION

OF R N A

IN C E L L E X T R A C T S

Cell extracts of E. coli B in o.I IV[ Tris ( p H 8.1) or in Tris buffer containing o.o 5 M E D T A were incubated for 3 ° rain at 37 ° in the presence of the concentrations of spermidine indicated in the table. Acid-soluble nucleotide products of the reaction were determined o n the perchloric acid supernatant fraction.

Spermidine

(raM)

o o.Io 0.25 0.50 I.OO 2.50 5.oo IO.OO 15.oo

Absorbance at 260 rot* o/acid-soluble/faction* Cell extract

Cell extract (/rozen)

Cell extract (0.05 M E D T A )

0.948 0,833 0.689 0.426 0.226 o.421 o. 704 o.818 o.847

0.928 0,899 0.898 0.849 o.787 0.860 o. 869 o.883 o.881

I,I88 I.o44 0,966 o.66o o.31o o.241 o. 15 ° 0.032 0.o05

" A v e r a g e a b s o r b a n c e of cold perchloric acid s u p e r n a t a n t fraction of replicate s a m p l e s .

polyamine causes a biphasic inhibition and reactivation of the enzymatic degradation of RNA in fresh cell extracts but is completely inactive when added to cell extracts previously frozen. In the presence of EDTA, the inhibition of the nuclease activity is comparable to inhibition in fresh preparations but, as with spermidine, the protection is complete and no reactivation occurs at any concentration of the polyamine.

II

TABLE EFFECT

OF SPERMINE

ON DEGRADATION

OF RNA

IN CELL EXTRACTS

C o n d i t i o n s of i n c u b a t i o n are t h o s e i n d i c a t e d in t h e legend of Table I.

Spermine

(raM)

o o.oi 0.02 o.04 o.05 o.Io o.25 o.5o I.OO 2.5 o 5.oo

Absorbance at 260 ink* o[ acid-soluble #action* Cell extract

Cell extract (/rozen)

Cell extract (0.05 M E D T A )

0.894 0.783 0.765 0,720 0.440 0.263 o.4ol 0.46o o.486 o.593 0.803

0.888 0,842 o.861 o.8o7 o.863 o.892 o.849 o.877 0.884 0.875 0,843

1.188 1.oo 5 1.o48 o.952 o,81o 0,767 o,643 0,437 0,298 0,023 0.0o5

" A v e r a g e a b s o r b a n c e of cold perchloric acid s u p e r n a t a n t fraction of replicate samples.

Biophys. Biophys. Acta, 157 (1968) 43-51

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47

EHect o] polyamines on the degradation o/ RNA in ribosomes Tile addition of either spermidine or spermine to ribosomes incubated at 37 ° in o.i M Tris buffer (pH 8.1) protected the ribosomes from enzymatic destruction and the protection was biphasic as in the experiments with cell extracts (Table III). TABLE

III

]EFFECT O F POLYAMINI~S O N D E G R A D A T I O N

OF RNA

IN RIBOSOMEES O F E. coil B

Fresh or frozen ribosomes were incubated in o. i IV[Tris ( p H 8.1) containing spermidine or spermine

(as indicated in t h e table). After 3 ° min at 37 ° the reaction w a s s t o p p e d w i t h cold perchloric a c i d - u r a n y l acetate. All absorbancies (A260 my) are the average absorbance of the cold perchloric acid s u p e r n a t a n t fractions of replicate samples.

Spermidine (raM)

Ribosomes (A26o ray)

Frozen ribosomes (A 260 m~)

Spermine (raM)

Ribosomes (A260 ra~)

Frozen ribosomes (A 260 m~*)

o 0.005 o.o 5 o. IO o.15 0.20 o.25 o.5o I.OO 5.oo IO.OO

1.181 1.171 1.155 I. I24 o.936 o.881 o.751 o.466 o.435 0.668 0.765

1.192 1.165 1.155 I. 138 o.977 o.868 0.77o 0.452 o.4ol 0.673 0.792

o O.OLO O.Ol 5 o.o2o o.o25 0.050 o.Io o.2o o.50 I.OO 5.00

1.281 o.882 0.662 0.473 o.419 0.376 0.570 0.582 o.547 0.639 0.756

1.199 o.925 0.7o0 o.498 0.422 0.349 0.487 o.569 o-575 o.650 0.77o

Ribosomes which were frozen and incubated on the following day could not be distinguished from fleshly prepared ribosomes, in contrast to frozen cell extracts which were not protected from enzymatic degradation by the polyalnines. Ribosomes incubated in Tris-EDTA supplemented with polyamines were not degraded by the characteristic biphasic enzyme activity associated with fresh and frozen ribosomes but, as in cell extracts, the polyamine inhibition was complete and irreversible. Evidence has been obtained which indicates that the ribosomal RNA protected from enzymatic degradation in the presence of polyamines remains intact. The RNA of ribosomes which had been incubated in the presence of I mM spermidine was separated from the incubation mixtures by the phenol procedure of KURLAND9. The aqueous solution of RNA was concentrated by lyophilization and centrifuged in a 5-2o ~o sucrose gradient for 16 h at 24 5o0 rev./min. The principal absorbance peaks at 260 m# corresponded to the 23-S and I6-S components of rRNA. The 260 m/zabsorbing material from ribosomes incubated in the absence of spermidine or in the presence of 8 mM spermidine, prepared and analyzed b y identical phenol and sucrose gradient procedures, consisted of low molecular weight fragments which remained near tile top of the gradient.

E[[ect o/polyamines on the degradation o/rRNA by ribonuclease I The enzyme activity inhibited by the polyamines is characteristic of the riboclease I of E. coli ribosomes investigated by SPAHR AND HOLLINGWORTHa. This enBiochim. Biophys. Acta, 157 (1968) 43-51

V.A. ERDMANN gt al.

48

zyme degrades ribonucleic acid in alkaline media (pH 7.5-8.5) of high ionic strength (I O.l-O.2) and is activated in bacterial cells, extracts of cells, or ribosomes by incubation in high ionic strength buffer and by incubation with EDTA. The chelating agent is presumed to release the enzyme and to labilize the substrate by the removal of Mg ~+. The inhibition of the enzyme activity by low concentrations of polyamines and the apparent reversal of the inhibition by higher concentrations of polyamines suggested an interaction between the polycations and rRNA which both stabilized and labilized the substrate to enzymatic attack. This possible mechanism was tested with rRNA prepared from purified ribosomes of E. coli (ref. 8) and ribonuclease I also isolated from E. coti ribosomes 3. Purified enzyme and substrate were incubated with levels of the polyamines which modified the enzymatic degradation of ribonucleic acid in ribosomes. Neither spermidine nor spermine were inhibitory (Table IV); high conTABLE

IV

THE INACTIVITY OF POLYAMINES AS INHIBITORS OF THE DEGRADATION OF r R N A BY RIBONUCLEASE I R e a c t i o n m i x t u r e s p r e p a r e d in d u p l i c a t e in a final v o l u m e of 1.2 m l of o . i M Tris buffe r (pH 8.1) c o n t a i n e d t h e following: I m g of r R N A , o . o i m g r i b o n u c l e a s e I a n d s p e r m i d i n e or s p e r m i n e as i n d i c a t e d in t h e t a b l e . A f t e r i n c u b a t i o n a t 37 ° for 3 ° rain t h e r e a c t i o n w a s s t o p p e d w i t h cold p e r c h l o r i c a c i d - u r a n y l a c e t a t e . All a b s o r b a n c i e s (A260 m~) are t h e a v e r a g e a b s o r b a n c e of t h e cold p e r c h l o r i c acid s u p e r n a t a n t f r a c t i o n of r e p l i c a t e samples .

Spermidine (mM)

RNA degradation

Spermine (mM)

(A 2~o ,.~)

(A 2oo ~,) o 0.005 O.OLO o.Io 0.50 i.oo io.o

0.844 0.905 0.834 0.822 o.895 0.899 o.686"

RNA degradation

o o.oox 0.005 O.OLO o.o5o o.io 0.5o

0.983 0.965 0.870 o.81o 0.932 0.878 o.183"

* P r e c i p i t a t e of n u c l e i c a c i d a p p e a r e d before i n c u b a t i o n .

centrations of spermidine (IO/,moles) and lower concentrations of spermine (o.5 to IO #moles) precipitated the rRNA but there was no evidence of the concentrationdependent inhibition and reactivation of the enzyme system previously observed with cell extracts and ribosomes. The effect of the polyamines appears to be demonstrable only with tile "native" substrate (ribosomes) and with the "nascent" ribonuclease I presumably associated with ribosomes as a result of secondary redistribution of the enzyme after release from a membrane site 12.

Identification o/the nuclease activity which is inhibited and activated by polyamines The enzymatic reaction was more fully characterized by chromatography of the products on Dowex-I-formate columns as described in METHODS. No nucleoside 5'-phosphates were detected in the trichloroacetic acid-soluble fraction of ribosomes incubated for 15o min at 37 ° in o.I M Tris buffer (pH 8.1) containing o.oi M EDTA. Biochim. Biophys. /Iota, 157 (1968) 43-51

POLYAMINES AND RIBOSOME DEGRADATION

49

The products were predominantly adenosine 3'-phosphate and uridine 3'-phosphate with smaller amounts of guanosine 3'-phosphate and cytidine 3'-phosphate. These results are in agreement with similar experiments reported by WADE18 who identified tile products of the autodegradation of E . coli ribosomes in the presence of EDTA as nucleoside 3'-phosphates. SPAHR AND HOLLINGWORTH3 also identified 3'-isomers as the nucleotide products of a total digest of rRNA by purified ribonuclease I. It is apparent, therefore, that the ribonuclease I is activated in ribosomes under the conditions we have employed and that the polyamines, spermine and spermidine, can completely inhibit the release of the "nascent" nuclease. The products of the degradation of ribosomes in o.I M Tris buffer containing 8 mM spermidine ( the "nuclease" is reactivated by 5-1o inM spermidine) were also examined by chromatography on a Dowex-I-formate column. The products were nucleoside 3'-phosphates and, as in the EDTA system, no nucleoside 5'-phosphates were detected; it appears, therefore, that the ribonuclease I activity associated with ribosomes is both inhibited and activated by polyamines. The nucleotides produced in the autodegradation of ribosomes were also identified by our modification of the paper chromatographic procedure of 13LESNER11 for the separation of nucleoside 3'- and nucleoside 5'-phosphates. Only nucleoside 3'-phosphates were found in the acid-soluble products from the incubation of ribosomes in o.I M Tris containing 8 mM spermidine. Tile samples from the o.I M Triso.oi M EDTA incubation mixtures contained predominantly nucleoside 3'-phosphates but a small region of ultraviolet quenching was also present near the sample origin (the nucleoside 5'-phosphate region) on these plates. This spot was eliminated on thin-layer chromatograms which contained aliquots of the same sample previously hydrolyzed with 0.3 M NaOH and all of the nucleotides appeared in the nucleoside 3'-phosphate region of the thin-layer plate. This product, convertible to nucleoside 3'-phosphates b y mild alkaline hydrolysis, is probably a mixture of oligonucleotides.

TABLE

V

FAILURE OF SPERMIDINE TO ACTIVATE "NUCLEASES" IN EXTRACTS OF RIBONUCLEASE I

E. coli

MRE6oo

LACKING

Conditions of incubation are identical to those described in the legends of Tables I and III (E. coliB cell extracts and ribosomes). All absorbancies (A260 m~) are the average absorbance of the cold perchloric acid supernatant fraction of replicate samples.

Spermidine (mM)

Cell extract

Spermidine (raM)

Ribosomes (A 2aOrot*)

o o.ooi o.oo5 O.OLO o.o5o o.ioo 0.250 o.5oo I.OOO 5.000 to.oo

0.057 0.028 o.o28 o.o33 0.025 0.053 0.086 0.089 0.057 o. 142 o.197

(A 2~o ,,~ ) o O.lO 0.25 o.5o I.OO 2.50 5.00 io.oo 15.oo 2o.oo 25.0o

0.355 0.273 o.149 o.123 o.123 o.iIZ 0.089 o. lO 7 o. I O l o. 123 o.132

Biochim. Biophys. Acta,

157

(1968)

43-51

5°

v . A . ERDMANN et

al.

As in the autodegradation system activated by spermidine there is no evidence, therefore, for nuclease activity other than the ribonuclease I reaction which yields nucleoside 3'-phosphate products.

Autodegradation o/ribosomes [rom E. coli M R E 6 o o Confirmation for the evidence favoring the participation of polyamines in the autodegradation of ribosomes by ribonuclease I was sought in experiments with cell extracts and ribosomes prepared from E. coli strain MRE6oo which lacks ribonuclease I activity. There was marginal degradation of nucleic acid in cell extracts prepared from this organism (Table V) but ribosomes were not degraded under the conditions previously demonstrated to promote extensive degradation of E. coli B ribosomes. The most significant observation in these experiments, however, was the failure of spermidine to activate a nuclease at those concentrations of polyamine which induced a considerable degradation of ribonucleic acid in the E. coli B systems which contain ribonuclease I.

DISCUSSION The inhibition by polyamines of the enzymatic degradation of rRNA is demonstrable under conditions which are known to release ribonuclease r from an inactive association with ribosomes i.e., by incubation in alkaline buffer of high ionic strength or in media containing EDTA. Polyamines do not inhibit a purified ribonuclease I - r R N A reaction, however, under the same conditions of incubation. The action of the polyamines, therefore, appears to depend upon the maintenance of a masked ribonuclease r-ribosome relationship. The inability of ribonuclease r to attack rRNA until the enzyme is released by a disorganization of the ribosomes is well established12; in our experiments, the ribosome structure is labilized by incubation in o.I M Tris buffer or in the same buffer containing o.o5 M EDTA. The polyamines prevent the alteration of ribosomal structure which is induced by Tris or by EDTA and the reduced activity or inactivity of ribonuclease I suggests that the enzyme is not released from the ribosomes so protected. The reactivation of ribonuclease I when the concentration of spermidine is increased ( i - i o mM) indicates that the polyamines can also induce ribosome instability in contrast to the stabilized ribosome conformation (with masked ribonuclease I) which is maintained by low (o.I to x raM) concentrations of polyamine. The reactivation of ribonuclease I by high concentrations of polyamine (and presumably the alteration of ribosome structure) does not occur when 0.o 5 M EDTA is present during the incubation in o.I M Tris. The interpretation of this observation is not clear but it would appear that the ribosome conformation stabilized by spermidine in the presence of EDTA is not susceptible to degradation by ribonuclease r. It is possible that the polyamine can induce a refolding of the ribosome structure in the absence of Mg*+ which is quite different than the normal ribosome conformation. Unfolding of ribosomes b y EDTA has been demonstrated by GESTELAND14 and only partial refolding of the ribosomes to particles having abnormally low sedimentation coefficients was obtained by dialyzing unfolded ribosomes against o.oi M Mg 2+. The Biochim. Biophys. Acta, 157 (1968) 43 51

POLYAMINES AND RIBOSOME DEGRADATION

51

polyalnines might produce a refolded ribosome conformation in the absence of M g *+ which is quite different than the ribosome conformation attacked by ribonuclease I.

ACKNOWLEDGEMENTS

Published with the approval of the Director of the New Hampshire Agricultural Experiment Station as Scientific Contribution No. 423. This investigation was supported by Contract Nonr-37IO(O2), NR-Io3-3I 7, Office of Naval Research, Department of the Navy and by Public Health Service Grant AI-o5397 from the National Institute of Allergy and Infectious Diseases. REFERENCES I 2 3 4 5 6 7 8

9 IO ii 12 13 14

E. J. HERBST AND B. P. DOCTOR, J. Biol. Chem., 234 (1959) I497, G. H. THOMAS, P h . D . t h e s i s U n i v e r s i t y of M a r y l a n d , I963. P" F. SPAHR AND B. R. HOLLINGWORTH, J. Biol. Chem., 236 (1961) 823. P. F. SI~ARR, J. Biol. Chem., 239 (1964) 3716. H. TABOR AND C. W. TABOR, Pharmacol. Rev., 16 (1964) 245, R_. B. ROBERTS, P. H, ABELSON, D. B. COWlE, E. T. BOLTON AND R. B. BRITTEN, Carnegie Inst. Wash. Publ., 607 (1957) 5. K. A. CAMMACK AND H. E. WADE, Bioehem. J., 96 (1965) 67I. A. TlSSI~RES, J. D. WATSON, D. SCHLESSINGER AND B. R. HOLLINGW'ORTH, J. Mol. Biol., (1959) 221. C. G. KURLAND, J. Mol. Biol., 2 (196o) 83. W. E. COHN, J. Am. Chem. Soe., 72 (195 ° ) 1471. P. PLESNER, Acta Chem. Seand., 9 (1955) 197H. C. NEU AND L. A. HEPPEL, Proc. Natl. Aead. Sci. U.S., 51 (1964) 1267. H. E. WADE, Bioehem. J., 78 (1961) 457. R. F. GESTELAND, J. Mol. Biol., 18 (1966) 356.

Biochim. Biophys. Acta, 157 (1968) 43-51