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RNA with high specific activity in neurons and glia
197
SHORT COMMUNICATIONS
clicks the a m p l i t u d e o f the responses d r o p s m a r k e d l y (Fig. 1, N C a ) . * Fig. 2 shows the p a t t e r n s o f the potentials in the c o c h l e a r nucleus, in the s u p e r i o r olive, in a u d i t o r y area I o f the cerebral cortex a n d in the c a u d a t e nucleus after s t i m u l a t i o n with clicks o f various intensities. There is a similar b e h a v i o u r o f the p o t e n t i a l s in the a u d i t o r y a r e a I a n d in the c a u d a t e nucleus, b o t h exhibiting an a l m o s t c o n s t a n t voltage response to stimulations f r o m - 2 0 to - 5 0 dB. I n o u r opinion, this suggests t h a t there is some functional c o r r e l a t i o n between these two structures. This w o r k was s u p p o r t e d by a g r a n t f r o m Consiglio N a z i o n a l e delle Ricerche, Italy. V. LA GRUTTA S. ABBADESSA
Istituto di Fisiologia umana dell'Universit~ di Palerme, Palermo, Italia
I ALBE-FESSARD,D., OSWALDO-CRuz, E., AND ROCHA-MIRANDA,C., Activit6s 6voqu6es dans le
2 3
4 5 6
noyau caud6 du chat en r6ponse ~t des types divers d'aff6rences, l~tude macrophysiologique, Electroenceph. clin. Neurophysiol., 12 (1960)405420. BONVALLET,M., DELL, P., AND HUGELIN,A., Projections olfactives, gustatives, visc6rales, vagales, visuelles et auditives au niveau des formations grises du cerveau ant6rieur du chat, J. Physiol. (Paris), 44 (1952) 222-224. DESMEDT, J. E., Auditory-evoked potentials from cochlea to cortex as influenced by activation of the efferent olivo-cochlear bundle, J. acoust. Soc. Amer., 34 (1962) 1478-1496. DESMEDT,J. E., AND FRANKEN, L., The retrotentorial part of the cat's brain stem in oblique stereotaxic coordinates, J. Physiol. (Lond.), 142 (1958) 15P-17P. LA GRUTTA, V., AND ABBADESSA,S., Studio elettrofisiologico del nucleo caudato. Nora preliminare, Boll. Soc. ital. Biol. sper., 41 0965) 1341-1343. SEGUNDO, J. P., AND MACHNE, X., Unitary responses to afferent volleys in lenticular nucleus and claustrum, J. Neurophysiol., 19 (1956) 325-339.
(Received March 28th, 1966) Brain Research, 2 (1966) 195-197
* As previously shown by Desmedt a, a similar pattern is found for auditory area I of the cerebral cortex (Fig. l, A1), whereas a more linear relation is observed in the cochlear nucleus (antero-ventral region) and the superior olive (Fig. 1, NCo and OS).
RNA with high specific activity in neurons and glia I n the study of h y b r i d f o r m a t i o n between D N A a n d i s o t o p i c a l l y labelled R N A , i n c u b a t i o n with ribonuclease in low c o n c e n t r a t i o n s has been used as one m e t h o d o f choice. By this t e c h n i q u e u n p a i r e d R N A is e l i m i n a t e d which is n o t involved in h y d r o g e n b o n d i n g a n d thus accessible to d e g r a d a t i o n with ribonuclease 6. W a r n e r et al. 5 presented the first b i o c h e m i c a l a n d m o r p h o l o g i c a l evidence t h a t the R N A sensitive to very low c o n c e n t r a t i o n s o f r i b o n u c l e a s e was single s t r a n d e d messenger R N A connecting ribosomes. F u r t h e r m o r e , this p o l y s o m a l structure was the site o f specific p r o t e i n p r o d u c t i o n . The D e i t e r s ' n e u r o n s used in the p r e s e n t study are c h a r a c t e r i z e d b o t h by p o l y Brain Research, 2 (1966) 197-200
198
SHORT COMMUNICATIONS
somes containing 3 to 6 ribosomes, but also by giant polysomes consisting of around 100 ribosomes 3. Recently, Egyhazi z has devised a method for fractionating R N A in single neurons and glia using phenol extraction at different temperatures. Treatment of the isolated, fresh cells with phenol at 3 ° left an RNA fraction which constituted around 5 0 ~ of the total RNA. By increasing the phenol temperature to 45 ° an additional small R N A fraction was released from nerve cells the base ratios of which was characterized by high adenine and uracil values. It is to be noted that the main part of the total nerve cell RNA is composed by cytoplasmic, ribosomal RNA. By contrast, the glial RNA is mainly nuclear R N A with the sampling procedure used. The aim with the present studies has been to combine the above described micro-fractionating method with incubation of the cells in low concentrations of ribonuclease. This was done in order to characterize the R N A fraction being released at phenol extraction between 3 ° and 45 °. Rabbits were given [3H]-orotic acid with a specific activity of 2.3 C/mMintraventricularly. Each rabbit received 150 #C in 150 #l of distilled water through a permanent cannula inserted into the 4th ventricle 4. Neurons and their surrounding glia were isolated from the lateral vestibular nucleus by free hand disseclion under a stereo-microscope at 80 times magnification. The dissection was carried out on a freezing microscope table which kept the temperature at around 0 °. The cells were treated for 1 h with 3 ° water saturated phenol which was removed by 95 ~ ethanol. After this stage, all procedures were carried out in an inverted paraffine oil chamber. The ribonuclease (EC 2.7.7.16.) concentrations used are given in Table I. All incubations were carried out at room temperature. It is to be remembered that in Lhe present experiments whole, single ceils were used. In pilot experiments it was first shown that the usual low concentration of 0.25 #g/ml did not give measurable fractions of RNA. This presumably depends on the fact that the cell membranes, at least partly, are maintained and that the penetration into the interior of the cell is unsatisfactory. Even the concentration of l0 #g of ribonuclease per ml gave too great a variation of the small fraction of RNA with high labelling. The determination of specific activity was carried out in the following way using the procedure of Koenig and Brattgglrd 4. First the amount of R N A in #/zg was determined in each fraction according to Edstrbm 1. This amount of R N A was placed as a microdrop on a 80 # wide cellophane strip and was dried. The part of the strip containing the R N A was introduced in a supramax glass capillary (Schott Co., GSttingen, Germany) together with particles of Zn and perchlorate and combusted at 650 ° for 45 min. This releases the tritium to gas and the activity is measured with high efficiency in a Geiger system. The results in Table I show that the highest specific activities of the R N A fractions are obtained with the lowest concentrations of ribonuclease. The specific activity of R N A increases with decreasing size of the R N A fraction being released at the 50 #g/ml range of ribonuclease. Each figure for specific activity represents one analysis of material from one animal. Brain Research, 2 (1966) 197-200
199
SHORT COMMUNICATIONS
TABLE I PHENOL--RIBONUCLEASEEXTRACTIONOF A HIGHLY-LABELLEDRNA FRACTIONFROMNEURONSAND GLIA Phenol extraction was performed at 3°. Ribonuclease was used as a 50 #g per ml water solution and as 400 #g per ml of 0.2 N ammonium acetate, respectively.
Ribonuclease 50 I~g/ml No. of Expts.
Ribonuclease 400 i~g/ml
% RNA released
% activity of RNA released
Specific activity c.p.m./izg
% RNA released
% activity of RNA released
1N 2N Neurons 3N 4N
11.4 4.8 0.6 1.1
50 35 25 51
310 285 2500 11,790
88.6 95.2 99.4 98.9
50 65 75 49
40 27 46 88
1G 2G 3G 4G
52.4 5.5 8.5 0.7
75 9 22 2
85 932 219 4950
47.6 94.5 91.5 99.3
25 9l 78 98
32 540 71 2270
Glia
Specific activity c.p.m.//~g
As is clearly seen f r o m the data in Tab l e I the small fraction o f R N A released during this p r o c e d u r e has a very high specific activity. A n increased release o f R N A results in a m a r k e d d r o p in specific activity. This leads to the conclusion that a very small R N A fraction exists in the n e r v e cell and has an exceedingly high specific activity. T o extract specifically this rapidly labelled R N A f r o m the n e u r o n is a q u e s t i o n o f a d a p t i n g time, t e m p e r a t u r e an d ribonuclease c o n c e n t r a t i o n (Fig. 1). T h e base c o m p o s i t i o n o f the R N A f r o m b o t h n e u r o n s an d glia at the l o w ribonuclease c o n c e n t r a t i o n was d e t e r m i n e d on the basis o f U V a b s o r b a n c e and has a decidedly lo w e r G q - C / A + U ratio (1.1; 1.0) c o m p a r e d to that o b t a i n e d at the
TABLE II BASERATIOCOMPOSITIONOF RNA FRACTIONSOBTAINEDBY DIFFERENTIATEDEXTRACTIONAND RIBONUCLEASEDIGESTION Acid hydrolysis by 4 NHCl at 100° for 30 rain, base ratios determined on the basis of UV absorbance.
Neurons
Adenine Guanine Cytosine Uradl
Glia
RNA 50 I~g/ml
RNA 400 I~g/ml RNA 50 I~g/ml
RNA 400 I~g/ml
22 25 27 26
21 30 32 17
20 31 30 19
23 27 24 26
G+C 1.1
1.6
1.0
1.6
A+U
Brain Research, 2 (1966) 197-200
200
SHORT COMMUNICATIONS
NEURONS Experiment
GLIA
I~lUl l il l l
[
1G
II/l[lllllll[lll[lllllllllllllllll[l!]llllllll[l[lllllllillll
J
2N
HLIIl
I
2G
I@
I
3N
[
J
3G
Ilml l l
I
4N
I[I ,
I
I i
,
,
,
o Io 2o 3o 4o
,
,
t
7o 86
io'o°I.
6 16 26 3b 46
76 B6 961o6'I,
Fig. 1. The ratios between the specific activity and the RNA released by the method used in 8 experiments are demonstrated, the values are given as percentages of the total RNA before degradation with ribonuclease. Hatched area: specific activity; striped area: RNA content. higher ribonuclease c o n c e n t r a t i o n . I n the latter case the G ÷ C / A + U ratio was 1.6, indicating a r i b o s o m a l R N A c h a r a c t e r (Table I1). T h e a t t e m p t to differentiate r a p i d l y - l a b e l l e d R N A by the present m e t h o d was m o r e successful in the case o f the n e u r o n s t h a n in t h a t o f the glia. The ratio o f specific activity o f the n e u r o n a l R N A released at low a n d at high ribonuclease conc e n t r a t i o n s was f o u n d to be m u c h higher t h a n the c o r r e s p o n d i n g r a t i o o f the glial RNA. A p r o b a b l e e x p l a n a t i o n could be t h a t the p h e n o l p r o c e d u r e used releases m o r e R N A f r o m the glial c y t o p l a s m . This will result in a p r e d o m i n a n t l y nuclear, r a p i d l y labelled R N A being released by the enzyme t r e a t m e n t o f the glia. I n the neurons, on the o t h e r h a n d , the c o r r e s p o n d i n g t r e a t m e n t releases a cytoplasmic R N A with a very high specific activity. This investigation was s u p p o r t e d b y the Swedish M e d i c a l R e s e a r c h Council a n d by U.S. A i r F o r c e E u r o p e a n Office o f A e r o s p a c e Research, G r a n t EOAR-63-28. Institute of Neurobiology, University of G6teborg, GOteborg (Sweden)
ENDREEGYHAZI HOLGER HYDEN
1 EDSTROM,J.-E., Microextraction and microelectrophoresis for determination and analysis of nucleic
acids in isolated cellular units. In D. M. PRESCOTT (Ed.), Methods in Cell Physiology, Vol. 1, Academic Press, New York, 1964, pp. 417-447. 2 EGYHAZI, E., Microchemical fractionation of neuronal and glial RNA, Biochim. biophys, acta, 114 (1966) 516-526. 3 EKHOLM, R., AND HYO~N, H., Polysomes from microdissected fresh neurons, J. ultrastruct. Res., 13 (1965) 269-280. 4 KOENIG, E., AND BRATTG~RD, S.-O., A quantitative micro method for determination of specific radioactivities of H3-purines and HZ-pyrimidines, Analyt. Biochem., 6 (1963) 424-434. 5 WARNER,J. R., KNOPF, P. M., AND RaCH, A., A multiple ribosomal structure in protein synthesis, Proc. nat. Acad. Sci. (Wash.), 49 (1963) 122-129. 6 YANKOFSKY,S. A., AND SP~EGELMAN,S., The identification of the ribosomal RNA cistron by sequence complementarity, I. Specificity of complex formation, Proc. nat. Acad. Sci. (Wash.), 48 (1962) 1069-1078. (Received May 21st, 1966) Brain Research, 2 (1966) 197-200
SHORT COMMUNICATIONS
clicks the a m p l i t u d e o f the responses d r o p s m a r k e d l y (Fig. 1, N C a ) . * Fig. 2 shows the p a t t e r n s o f the potentials in the c o c h l e a r nucleus, in the s u p e r i o r olive, in a u d i t o r y area I o f the cerebral cortex a n d in the c a u d a t e nucleus after s t i m u l a t i o n with clicks o f various intensities. There is a similar b e h a v i o u r o f the p o t e n t i a l s in the a u d i t o r y a r e a I a n d in the c a u d a t e nucleus, b o t h exhibiting an a l m o s t c o n s t a n t voltage response to stimulations f r o m - 2 0 to - 5 0 dB. I n o u r opinion, this suggests t h a t there is some functional c o r r e l a t i o n between these two structures. This w o r k was s u p p o r t e d by a g r a n t f r o m Consiglio N a z i o n a l e delle Ricerche, Italy. V. LA GRUTTA S. ABBADESSA
Istituto di Fisiologia umana dell'Universit~ di Palerme, Palermo, Italia
I ALBE-FESSARD,D., OSWALDO-CRuz, E., AND ROCHA-MIRANDA,C., Activit6s 6voqu6es dans le
2 3
4 5 6
noyau caud6 du chat en r6ponse ~t des types divers d'aff6rences, l~tude macrophysiologique, Electroenceph. clin. Neurophysiol., 12 (1960)405420. BONVALLET,M., DELL, P., AND HUGELIN,A., Projections olfactives, gustatives, visc6rales, vagales, visuelles et auditives au niveau des formations grises du cerveau ant6rieur du chat, J. Physiol. (Paris), 44 (1952) 222-224. DESMEDT, J. E., Auditory-evoked potentials from cochlea to cortex as influenced by activation of the efferent olivo-cochlear bundle, J. acoust. Soc. Amer., 34 (1962) 1478-1496. DESMEDT,J. E., AND FRANKEN, L., The retrotentorial part of the cat's brain stem in oblique stereotaxic coordinates, J. Physiol. (Lond.), 142 (1958) 15P-17P. LA GRUTTA, V., AND ABBADESSA,S., Studio elettrofisiologico del nucleo caudato. Nora preliminare, Boll. Soc. ital. Biol. sper., 41 0965) 1341-1343. SEGUNDO, J. P., AND MACHNE, X., Unitary responses to afferent volleys in lenticular nucleus and claustrum, J. Neurophysiol., 19 (1956) 325-339.
(Received March 28th, 1966) Brain Research, 2 (1966) 195-197
* As previously shown by Desmedt a, a similar pattern is found for auditory area I of the cerebral cortex (Fig. l, A1), whereas a more linear relation is observed in the cochlear nucleus (antero-ventral region) and the superior olive (Fig. 1, NCo and OS).
RNA with high specific activity in neurons and glia I n the study of h y b r i d f o r m a t i o n between D N A a n d i s o t o p i c a l l y labelled R N A , i n c u b a t i o n with ribonuclease in low c o n c e n t r a t i o n s has been used as one m e t h o d o f choice. By this t e c h n i q u e u n p a i r e d R N A is e l i m i n a t e d which is n o t involved in h y d r o g e n b o n d i n g a n d thus accessible to d e g r a d a t i o n with ribonuclease 6. W a r n e r et al. 5 presented the first b i o c h e m i c a l a n d m o r p h o l o g i c a l evidence t h a t the R N A sensitive to very low c o n c e n t r a t i o n s o f r i b o n u c l e a s e was single s t r a n d e d messenger R N A connecting ribosomes. F u r t h e r m o r e , this p o l y s o m a l structure was the site o f specific p r o t e i n p r o d u c t i o n . The D e i t e r s ' n e u r o n s used in the p r e s e n t study are c h a r a c t e r i z e d b o t h by p o l y Brain Research, 2 (1966) 197-200
198
SHORT COMMUNICATIONS
somes containing 3 to 6 ribosomes, but also by giant polysomes consisting of around 100 ribosomes 3. Recently, Egyhazi z has devised a method for fractionating R N A in single neurons and glia using phenol extraction at different temperatures. Treatment of the isolated, fresh cells with phenol at 3 ° left an RNA fraction which constituted around 5 0 ~ of the total RNA. By increasing the phenol temperature to 45 ° an additional small R N A fraction was released from nerve cells the base ratios of which was characterized by high adenine and uracil values. It is to be noted that the main part of the total nerve cell RNA is composed by cytoplasmic, ribosomal RNA. By contrast, the glial RNA is mainly nuclear R N A with the sampling procedure used. The aim with the present studies has been to combine the above described micro-fractionating method with incubation of the cells in low concentrations of ribonuclease. This was done in order to characterize the R N A fraction being released at phenol extraction between 3 ° and 45 °. Rabbits were given [3H]-orotic acid with a specific activity of 2.3 C/mMintraventricularly. Each rabbit received 150 #C in 150 #l of distilled water through a permanent cannula inserted into the 4th ventricle 4. Neurons and their surrounding glia were isolated from the lateral vestibular nucleus by free hand disseclion under a stereo-microscope at 80 times magnification. The dissection was carried out on a freezing microscope table which kept the temperature at around 0 °. The cells were treated for 1 h with 3 ° water saturated phenol which was removed by 95 ~ ethanol. After this stage, all procedures were carried out in an inverted paraffine oil chamber. The ribonuclease (EC 2.7.7.16.) concentrations used are given in Table I. All incubations were carried out at room temperature. It is to be remembered that in Lhe present experiments whole, single ceils were used. In pilot experiments it was first shown that the usual low concentration of 0.25 #g/ml did not give measurable fractions of RNA. This presumably depends on the fact that the cell membranes, at least partly, are maintained and that the penetration into the interior of the cell is unsatisfactory. Even the concentration of l0 #g of ribonuclease per ml gave too great a variation of the small fraction of RNA with high labelling. The determination of specific activity was carried out in the following way using the procedure of Koenig and Brattgglrd 4. First the amount of R N A in #/zg was determined in each fraction according to Edstrbm 1. This amount of R N A was placed as a microdrop on a 80 # wide cellophane strip and was dried. The part of the strip containing the R N A was introduced in a supramax glass capillary (Schott Co., GSttingen, Germany) together with particles of Zn and perchlorate and combusted at 650 ° for 45 min. This releases the tritium to gas and the activity is measured with high efficiency in a Geiger system. The results in Table I show that the highest specific activities of the R N A fractions are obtained with the lowest concentrations of ribonuclease. The specific activity of R N A increases with decreasing size of the R N A fraction being released at the 50 #g/ml range of ribonuclease. Each figure for specific activity represents one analysis of material from one animal. Brain Research, 2 (1966) 197-200
199
SHORT COMMUNICATIONS
TABLE I PHENOL--RIBONUCLEASEEXTRACTIONOF A HIGHLY-LABELLEDRNA FRACTIONFROMNEURONSAND GLIA Phenol extraction was performed at 3°. Ribonuclease was used as a 50 #g per ml water solution and as 400 #g per ml of 0.2 N ammonium acetate, respectively.
Ribonuclease 50 I~g/ml No. of Expts.
Ribonuclease 400 i~g/ml
% RNA released
% activity of RNA released
Specific activity c.p.m./izg
% RNA released
% activity of RNA released
1N 2N Neurons 3N 4N
11.4 4.8 0.6 1.1
50 35 25 51
310 285 2500 11,790
88.6 95.2 99.4 98.9
50 65 75 49
40 27 46 88
1G 2G 3G 4G
52.4 5.5 8.5 0.7
75 9 22 2
85 932 219 4950
47.6 94.5 91.5 99.3
25 9l 78 98
32 540 71 2270
Glia
Specific activity c.p.m.//~g
As is clearly seen f r o m the data in Tab l e I the small fraction o f R N A released during this p r o c e d u r e has a very high specific activity. A n increased release o f R N A results in a m a r k e d d r o p in specific activity. This leads to the conclusion that a very small R N A fraction exists in the n e r v e cell and has an exceedingly high specific activity. T o extract specifically this rapidly labelled R N A f r o m the n e u r o n is a q u e s t i o n o f a d a p t i n g time, t e m p e r a t u r e an d ribonuclease c o n c e n t r a t i o n (Fig. 1). T h e base c o m p o s i t i o n o f the R N A f r o m b o t h n e u r o n s an d glia at the l o w ribonuclease c o n c e n t r a t i o n was d e t e r m i n e d on the basis o f U V a b s o r b a n c e and has a decidedly lo w e r G q - C / A + U ratio (1.1; 1.0) c o m p a r e d to that o b t a i n e d at the
TABLE II BASERATIOCOMPOSITIONOF RNA FRACTIONSOBTAINEDBY DIFFERENTIATEDEXTRACTIONAND RIBONUCLEASEDIGESTION Acid hydrolysis by 4 NHCl at 100° for 30 rain, base ratios determined on the basis of UV absorbance.
Neurons
Adenine Guanine Cytosine Uradl
Glia
RNA 50 I~g/ml
RNA 400 I~g/ml RNA 50 I~g/ml
RNA 400 I~g/ml
22 25 27 26
21 30 32 17
20 31 30 19
23 27 24 26
G+C 1.1
1.6
1.0
1.6
A+U
Brain Research, 2 (1966) 197-200
200
SHORT COMMUNICATIONS
NEURONS Experiment
GLIA
I~lUl l il l l
[
1G
II/l[lllllll[lll[lllllllllllllllll[l!]llllllll[l[lllllllillll
J
2N
HLIIl
I
2G
I@
I
3N
[
J
3G
Ilml l l
I
4N
I[I ,
I
I i
,
,
,
o Io 2o 3o 4o
,
,
t
7o 86
io'o°I.
6 16 26 3b 46
76 B6 961o6'I,
Fig. 1. The ratios between the specific activity and the RNA released by the method used in 8 experiments are demonstrated, the values are given as percentages of the total RNA before degradation with ribonuclease. Hatched area: specific activity; striped area: RNA content. higher ribonuclease c o n c e n t r a t i o n . I n the latter case the G ÷ C / A + U ratio was 1.6, indicating a r i b o s o m a l R N A c h a r a c t e r (Table I1). T h e a t t e m p t to differentiate r a p i d l y - l a b e l l e d R N A by the present m e t h o d was m o r e successful in the case o f the n e u r o n s t h a n in t h a t o f the glia. The ratio o f specific activity o f the n e u r o n a l R N A released at low a n d at high ribonuclease conc e n t r a t i o n s was f o u n d to be m u c h higher t h a n the c o r r e s p o n d i n g r a t i o o f the glial RNA. A p r o b a b l e e x p l a n a t i o n could be t h a t the p h e n o l p r o c e d u r e used releases m o r e R N A f r o m the glial c y t o p l a s m . This will result in a p r e d o m i n a n t l y nuclear, r a p i d l y labelled R N A being released by the enzyme t r e a t m e n t o f the glia. I n the neurons, on the o t h e r h a n d , the c o r r e s p o n d i n g t r e a t m e n t releases a cytoplasmic R N A with a very high specific activity. This investigation was s u p p o r t e d b y the Swedish M e d i c a l R e s e a r c h Council a n d by U.S. A i r F o r c e E u r o p e a n Office o f A e r o s p a c e Research, G r a n t EOAR-63-28. Institute of Neurobiology, University of G6teborg, GOteborg (Sweden)
ENDREEGYHAZI HOLGER HYDEN
1 EDSTROM,J.-E., Microextraction and microelectrophoresis for determination and analysis of nucleic
acids in isolated cellular units. In D. M. PRESCOTT (Ed.), Methods in Cell Physiology, Vol. 1, Academic Press, New York, 1964, pp. 417-447. 2 EGYHAZI, E., Microchemical fractionation of neuronal and glial RNA, Biochim. biophys, acta, 114 (1966) 516-526. 3 EKHOLM, R., AND HYO~N, H., Polysomes from microdissected fresh neurons, J. ultrastruct. Res., 13 (1965) 269-280. 4 KOENIG, E., AND BRATTG~RD, S.-O., A quantitative micro method for determination of specific radioactivities of H3-purines and HZ-pyrimidines, Analyt. Biochem., 6 (1963) 424-434. 5 WARNER,J. R., KNOPF, P. M., AND RaCH, A., A multiple ribosomal structure in protein synthesis, Proc. nat. Acad. Sci. (Wash.), 49 (1963) 122-129. 6 YANKOFSKY,S. A., AND SP~EGELMAN,S., The identification of the ribosomal RNA cistron by sequence complementarity, I. Specificity of complex formation, Proc. nat. Acad. Sci. (Wash.), 48 (1962) 1069-1078. (Received May 21st, 1966) Brain Research, 2 (1966) 197-200