SHORT COMMUNICATIONS 157

x E. SCARANO, Biochim. Biophys. ACRE, 29 (1958) 459. 2 E. SCARANO, J. Biol. Chem., 235 (i96o) 706. s G. F. MALEY AND F. IViALEY, J. Biol. Chem., 234 (1959) 2975. 4 F. MALE'," aND G. F. MALEY, J. Biol. Chem., 235 (196o) 2968. b D. K. MYERS, C. A. HEMPHILL AND C. ~V~. TOWNSEND, Can. J. Biochem. Physiol., 39 (1961) IO 4. * E. SCARANO, L. BONADUCE AND B. DE PETROCELLIS, J. Biol. Chem., 237 (1962) 3342. ~' S. FIALA, A.. FIALA AND N. G'LINSMAN, Pathol. Biol., 9 (1961) 613. 8 F. MALEY AND G. F. MALEY, Cancer Res., 21 (1961) 1421. 9 S. FIhL& AND A. FIALA, Biochim. Biophys, Acta, 49 (1961) 228.

x0 M. TAL&EICO, R. CERRA, G. CAPUTO AND E. SCARANO, Bull. Soc. Ital. Biol. Sperim., 36 (196o) 1449.

11 G. F. MALEY AND F. MALI~Y, ]. Biol. Chem., 237 (1962) PC 3311. 12 j. PLIML, J. KARA AND F. ~ORM, Collection Czechosl. Chem. Commun., in the press. x3 j. PLIML ANn F. ~ORM, Collection Czechosl. Chem. Commun., 28 (1963) 546, 14 G. M. TENER, J. Am, Chem. Soc., 83 (196I) 159. 15 B. M. MAGASANIK, E. VISCHER, ~. DONIGER, D. ELSON AND E. CHARGAFF, J. Biol. Chem.,

186 (195o) 37. xe j . KkRA, unpublished results. aT j . BER£NEK AND F. ~ORr~, Collection Czechosl. Chem. Commun., 28 (I963) 459. xs j . C. GEEHARD Ar~I) A. B. 7?ARDEE, J. Biol. Chem., 237 (I962) 891. xD 1~. E. HANDSCHUiACHER ̂ ND C. A. PASTERNAK, Biochim. Biophys. AcrE, 3 ° (1958) 451- s0 j. ~KODA, J. K£EA, Z. ~ORiOVA ANO F. ~ORi, Biochim. Biophys. AcrE, 33 (1959) 579. zx E. SCARANO, G. GERACI, A. POLZELLA AND E. CAMPANILE, J. Biol. Chem., 238 (1963) PC 1556.

Received J u l y I6th, 1963

Biochim. Biophys. AcrE, 80 (1964) i54-i57

SC 712.1

18-S and 30-S fractions from the R N A of Krebs-2 ascites cells with differing base composition

RNA, extracted with phenol from whole Krebs-2 mouse ascites cells, consists of three main components. The smallest has a sedimenta t ion coefficient of 5-7 S, as measured in the analyt ical centrifuge1; most of it is soluble in I M NaC1 and corre- sponds to the t ransfer 1LNA. The two other components come presumably from ribosomes, by analogy wi th other systems, and have sedimenta t ion coefficients of, respectively, 16- I8 S and 28-30 S in solutions of moderate ionic strength1, 4.

The three peaks can be identif ied and separated by zone centr i fugat ion on a sucrose gradient. The sedimenta t ion coefficients of single-stranded R N A molecules are s t rongly dependent on ionic s t rength ' , as well as on the presence of d ivalent cations, par t icular ly magnes ium s . Thus it has been found tha t much of the clearest separation of the three components on zone centr ifugation can be achieved if all the procedures involved in extract ing and subsequent ly centrifuging the tLNA are carried out in the presence of E D T A (disodium salt Analar) at sufficient ionic strength. Removal of d ivalent ions from lZNA solutions with ion-exchange resins 3, or by E D T A in th i s way el iminates magnesium-coupled dimers and larger aggregates, and selects fairly uniform populat ions of molecules with narrowly defined ranges of molecular weights. However, such t r ea tmen t does not affect the p r imary s t ructure of RNA, since highly infectious lZ_RIA can be extracted from EMC virus by the same method 4.

Ext rac t ion of RNA_ from Krebs-2 cells on a large scale was performed by sus- pending I . lO 9 cells, previously washed in phosphate buffer-sal ine, in IOO ml of the

Biochim. Biophys. Acta, 80 (1964) I57--I60

I5~ Sft()RT COMMUNICATI()NS

following mixture: acetate buffer, o.oi M (pH 6.o) containing i 'i',, of bentonitea: 5o ml; EDTA (disodium salt), o.I M (pH 7.o):5o ml, and subsequently shaking this suspension with an equal volume of freshly distilled phenol ~aturated with water containing o.o 5 M EDTA for 15 rain at room temperature. Phenol extraction was repeated twice, and the aqueous layer was then precipitated 3 times with 2 w)l. of ethanol. ]nhe final precipitate was resuspended in the same buffer EDTA solution as that used for extraction, but this time in the presence of bentonite at a lower con- centration (o.I °~o). The latter was then removed by centrifuging for 3o min at 25 ooo rev./min and the supernatant stored at 4 ° in the presence of fresh bentonite (o.I °/o ) until ultracentrifugal analysis could be performed. Storage at + 4 ° under these conditions for several months did not affect the sedimentation properties of any of the three RNA components.

The reagents used for sucrose gradient centrifugation also contained EDTA at a concentration of o.o25 M, Tris-HC1 o.oo5 M (pH 7.2). Fractionation of the I8-S and 3o-S components was performed by layering I ml of a solution containing 2 mg of RNA on the top of a sucrose gradient (IO-2O ~/o sucrose), followed by centrifuging in the SW25 Spinco rotor for 14 h at 25 ooo rev./min at 5-1o °. At the end of this time approx. 4 ° fractions were then collected by piercing the bottom of the tube. Fig. I shows the profile of the successive fractions as given by absorbancy readings at 26o mff. The group of samples included in each separate peak were then pooled and

>- C) z < m

o

<

0.15

0.1

0.05

0

0

30 S

I 10

18S

I I I 20 30 40

FRACTION NUMBER

Fig. I. Sedimentat ion in a sucrose gradient of IZNA from Krebs-2 (see conditions in text).

reprecipitated with ethanol. Control of the purity of the fractionation was performed by centrifuging in sucrose gradient one aliquot of each pool; 85-9 ° % of ultraviolet- absorbing material sedimented as a single component.

Studies of changes in ultraviolet absorbancy at 260 m# in relation to heating suggested that differences in base composition between the two components might occur, since it is generally accepted that in RNA, as in I)NA, such changes can be referred to the melting of hydrogen bonds between complementary bases 6.

Samples of pure I8-S and pure 3o-S components were freed from EDTA by alcohol precipitation and resuspended in citrate-saline (NaC1, o. i5M; sodium

Biochim. Biophys. .4cta, 80 (1964) I57-10o

SHORT COMMUNICATIONS 159

citrate, o.oi5 M; pH 7.o) at a concentration adjusted to give an absorbancy of o.I at 260 m#. Separate samples were slowly heated in electrically heated silica cells in a Unicam Spectrophotometer SP 500. The temperature inside both the blank and the cells containing RNA were measured by small thermistors immersed in the solutions, using the original device of Vlzoso et al. ~. No correction was made for cell dilation or liquid expansion.

The ultraviolet absorbancy of either I8-S and 3o-S RNA changes gradually with rising temperature (Fig. 2), as already described by other workers for total

1.0 ,.~--o-.-o

<

< 0 .9 S "

~ o° °..o.°

~ 0 . 8

20 40 60 80 100 TEMPERATURE

Fig. 2. Re la t ive a b s o r b a n c y ~lAm~X~50mmtA ~60m~ of t he I8-S ( O - - - O ) and 3o-S ( Q - O ) c o m p o n e n t s as a func t ion of t e m p e r a t u r e in o.15 M NaCl -o .o I 5 M sod ium ci t ra te .

ribosomal RNA from different sourcese, s. The curves are almost completely reversible on fast or slow cooling, suggesting that, in both cases, the bonds concerned are rather intramolecular than between two chains. The maximum increase of absorption is 2 4 4 - 1 % of the initial value. Owing to the gradual transition in absorbancy, it is difficult to determine a precise melting temperature as with DNA. However, if we define as melting temperature (Tin) that temperature at which 50 % of the maximal increase has occurred, the Tm of the 3o-S component is nearly IO ° above the Tm of the I8-S component (Fig. 2); this suggests that in the 3o-S component there is more guanine and cytosine involved in base pairing than in the I8-S component, and possibly more of these two bases in the whole chain. This latter suggestion was later confirmed by base-ratio determinations. For these determinations, I8-S and 3o-S RNA isolated from several different gradients were used.

The RNA was hydrolysed to the nucleotides by incubation in 0.3 N KOH for 18 h at 37 ° (ref. 9). The alkaline digest was adjusted to p H 4.0 with HC104 and the insoluble KC104 centrifuged down. The supernatant was subjected to paper electro- phoresis l°, using 0.05 M ammonium acetate buffer. The electrophoresis papers were dried, the nucleotides located by examination in ultraviolet light, and eluted with o.oi N HC1. The base ratios were then calculated from the extinction at 260 m# using the molar extinction values given by BEAVEN et al. n .

The molar proportions of each nucleotide were then calculated on the basis of the mean z I. At least five determinations were made on each sample.

As read from the area of each peak, the 3o-S RNA represents approx. 60 %, and the I8-S RNA approx. 30 % of the total cell RNA. The calculated overall base ratios of the 3o-S and I8-S RbTA combined in a ratio of 2 : I agree well with the oh-

Biochim. Biophys. Acta, 8o II964) I57- I6O

160 SHORT COMMUNICATIONS

served base ratio of the total Krebs-cell RNA (Table I). Transfer RN'A may account for the slight difference observed.

T A B L E I

BASE RATIOS OF I8 -S RNA, 3o-S lZNA AND TOTAL KREBS-CELL IZNA"

Uridyl ic acid Guanyl ic acid Adeny l i c acid Cyt idy l ic acid

Tota l Calculated base x8-s R N A 30 s R N A Krebs-cel l ratio o/ combined

R N A 3 o - S + J c S - S R N A in propor t ion 2 : z

0.86 ±0 .02 0.68 ± o . o i 0.73 i o . o 2 o.74 I . I8 -~O.O2 1 . 4 4 ± o . o 4 1.31 ~ o . o 3 1.35 0.85 ±0 .02 0.64 2Lo.o 3 0.75 ~o .o2 o.73 1.12 ± 0 . 0 2 1.22 ±0 .03 1.21 ~o .o2 1.19

* Values are g iven ~= S.D.

Thus, in Krebs-2 cells the high guanine/cytosine content observed in ribosomal RNA is mainly a property of the large 3o-S component.

We thank Dr. F. K. SANDERS and Dr. A. T. H. BURNESS for their interest and advice on this work. One of us (A.B.) wishes to thank the Medical Research Council for a grant to support this work.

Virus Research Unit, Medical Research Council Laboratories,

Carshalton, Surrey (Great Britain)

L . MONTAGNIER

ANGELA D. BELLAMY

1 A. T. H. BURNESS AND A. D. VIZOSO, Biochim. Biophys. Acla, 49 (1961) 225. 2 H. BOEDTKER, J . Mol. Biol., 2 (196o) 171. 3 W. M6LLER AND H. BOEDTKER, in Acides ribonucldiques et polyphosphates, Colloque, C.N.t/ .S.

Par is , 1962, p. 99- 4 L. MONTAGNIER AND F. K. SANDERS, Nature, 197 (1963) 1178. 5 H. FRAENKEL-CONRAT, B. SINGER AND A. TSUGITA, Virology, 14 (1961) 54- 6 p . DOTY, ]-I. ~3OEDTKER, J. R. FRESCO, B. D. HALL AND a . HASELKORN, Ann. N .Y . Acad.

Sci., 81 (1959) 693. 7 A. D. VIzoso, A. T. H. BURNESS AND a . C. EMERY, in p repa ra t ion . s Vq. DINGMAN AND M. B. SPORN, Biochim. Biophys. Acta, 61 (1962) 164. 9 j . N. DAVIDSON AND R. M. S. SMELLIE, Biochem. J., 52 (1952) 594-

10 1~.. MARKHAM AND J. D. SMITH, Biochem. J., 52 (1952) 552- 11 Cv. H . I~EAVEN, E. R. HOLIDAY AND E . A. JOHNSON, in E. CHARGAFF AND J. N. DAVIDSON,

The Nucleic Acids, Vol. I, A c a d e m i c Press, New York, 1955, P. 493-

Received July 26th, 1963

Biochim. Biophys. Acta, 80 (1964) 157-16o

SC 7116

Streptomycin-sensitive and resistant ribosomes of Diplococcus pneumoniae

SPEYER et al. 1 and FLAKS et al. 2 clearly demonstrated the ribosomal localization of streptomycin sensitivity in cell-free systems obtained from Escherichia coli. In analogous experiments by the present authors the ribosomal localization was also found in a Diplococcus pneumoniae system; and some trials by immunological methods

Biochim. Biophys. Acta, 80 (1964) 16o-163