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Messenger ribonucleic acid activity in rabbit reticulocyte ribosomes
080
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.)
G. P.
GEORGIEV
M.I.
LERMAN
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
681
PRELIMINARY NOTES
The cell-free system employed to assay the RNA for messenger activity was basically that of IXTIRENBERG AND MATTHAE116.The RNA to be assayed was added to this system and incubated at 37 ° for 20 rain. The reaction was terminated by the addition of I ml of cold water and 3 ml of 12 % trichloroacetic acid. After standing at 4 ° for at least an hour the precipitate was dissolved in I ml of 0.5 N NaOH and heated at 37 ° for 45 rain. The protein was precipitated by the addition of 4 ml of 12 % trichloroacetic acid, the volume made up to 12 ml using a 7 % solution of this acid and the precipitate collected on a o.45-ff millipore filter. The filters were washed twice with 2 ml of 7 % trichloroacetic acid and glued to the back of aluminum counting planchets. They were dried by standing at room temperature overnight and counted in a Nuclear Chicago gas flow counter with a micromil window. When increasing amounts of reticulocyte ribosomal RNA were added to the E. coli cell-free system there was a corresponding increase in the amount of uniformly
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10 20 30 Fraction number'
Fig. I. Effect of reticulocyte ribosomal RNA concentration on uniformly 14C-labeled amino acid incorporation. The messenger RNA assay system (I.O ml) contained: 1. 4 mg of preincubated $30 protein16; o.5 mg of transfer RNA (both $30 and transfer RNA were from E. coli strain ATCC 8739); o. I 5 # C uniformly ~4C-labeled amino acids (1.38 mC/mg) obtained from New England Nuclear (Chlorella pyrenoidosa algal protein hydrolysate); 0.075 #mole of unlabelled glutamine, cysteine, tryptophan, methionine and asparagine; ioo/*moles Tris-HCI buffer (pH 7.8 at 20°); IO/,moles MgC12; 7 °/,moles KC1; 6/,moles fl-mercaptoethanol; I/,mole ATP; 6/,moles creatine phosphate; o.o3/,mole GTP; 24/,g creatine kinase (EC 2.7.3.2); plus the RNA to be assayed. Messenger RNA activity data are given as counts/min in total protein of the assay system over the control (minus reticulocyte RNA) value of lO25 counts/min. Fig. 2. Sedimentation profile obtained b y sucrose density gradient centrifugation of RNA prepared by phenol extraction. A 6.3-mg sample of ribosomal RNA was layered onto a 28 ml 4-20 % (w/v) linear sucrose gradient in o.o4 M Tris-HCl buffer (pH 7.3). The sucrose solution was previously treated with bentonite (3 mg/ml). The gradient was run at 24 ooo rev./min for 17 h in the Spinco Model L Ultracentrifuge using the SW 25.1 swinging bucket rotor. Fractions were obtained by puncturing the b o t t o m of the tube with a 2o-gauge needle and collecting drops. For assay, 0.25 ml of each fraction was added to the assay system. Messenger RNA activity data ( O - O ) are given as counts/min in total protein of the assay system over the control assay containing 0.25 ml of the appropriate sucrose solution. Absorbancy at 260 m/* ( O - - - O ) .
Biochim. Biophys..4cta, 91 (1964) 680-683
6S2
PRELIMINARY NOTES
~4C-labeled amino acids incorporated into the trichloroacetic acid precipitate. This increase is directly proportional to the amount of RNA added up to about o.5 mg (Fig. i). The incorporation was dependent upon ribosomes and an energy source and was inhibited by puromycin (Table I). TABLE I PROPERTIES
OF
THE
ASSAY
SYSTEM
The complete messenger R N A assay s y s t e m (see legend to Fig. i) was used with and w i t h o u t 0. 5 mg of reticulocyte ribosomal RNA, D a t a are given as c o u n t s / m i n in the total protein of the assay. Activity (counts[minlassay)
Assay conditions
Without RNA
with RNA
iooo ,235 257 265 25 °
.3900 235 298 285 245
Complete s y s t e m precipitated at zero time plus o . o 7 # m o l e puromycin minus ribosomes m i n u s energy* ATP, creatine phosphate, creatine kinase.
To determine if the messenger activity was found in a specific RNA fraction the reticulocyte RNA was fractionated by sucrose density gradient centrifugation and the fractions assayed for messenger activity. As shown in Fig. 2, the messenger activity is not equally distributed among the RNA's but is located in a fraction which migrates just behind the I7-S RNA. To help eliminate the possibility that the active fraction might result from RNA degradation during the phenol isolation, reticulocyte ribosomal RNA was prepared by the guanidine hydrochloridO 7 and lithium chloride 14methods. Both these methods yielded RNA preparations whose messenger RNA activity migrated in an identical position to that found for the messenger RNA activity of the phenol-isolated RNA. Although the method of isolation of the RNA is not critical, the presence of bentonite is and may account for the inability of other workers to fractionate the messenger RNA activity. If bentonite is not present in the isolation, variable activities are obtained. It should be noted that the peak of the messenger RNA activity consistently migrates slightly behind the absorbance peak of the small ribosomal RNA. This unequal distribution suggests an incomplete identity with the small ribosomal RNA. In fact, preliminary experiments show that the small ribosomal RNA peak can be separated into two fractions by methylated albumin chromatography. The demonstration of an RNA fraction which enhances amino acid incorporation into protein in a cell-free system, however, does not prove that one is dealing with a hemoglobin specific messenger RNA. In fact SHAEFFERet a/.lahave presented evidence against the synthesis of hemoglobin in an E . colt system when reticulocyte RNA is added. In contrast KRUH et al. ~ have shown that adding reticulocyte RNA to a reticulocyte cell-free system increases the synthesis of hemoglobin. The question of whether the RNA fraction reported in this communication is specific for hemoglobin must therefore await further studies. Biochim.
Bit)phys. Acla, 9x (1964) 680-683
683
PRELIMINARY NOTES
This investigation was supported by Public Health Service grant GM-Io999 from the National Institute of General Medical Sciences and General Research Support Grants FR-SoI-o518-oI and Soi-FR-o54o8oi.
Department o/ Biological Chemistry, University o/ Cincinnati Collegeo/Medicine, Cincinnati, Ohio (U.S.A.)
J O H N C. DRACH JERRY
B.
LINGREL
Biochem. Biophys. Res. Commun., 8 (1962) 9. D. NATHANS, G. VON EHRENSTEIN, R. MONRO AND V. LIPMANN, Federation Proc., 21 (1962) 127. j . BISHOP, G. FAVELUKES, l°t. SCHWEET AND E. RUSSELL, Nature, 191 (1961) 1365. j. ]3ISHOP AND R. SCHWEET, Biochim. Biophys. Acta, 65 (1962) 553. j . KRUH AND H. ]3ORSOOK, J. Biol. Chem., 220 (1956) 905. H. R. V. ARNSTEIN, R. A. COX AND J. A. HUNT, Nature, 194 (1962) lO42. H. ~_. V. ARNSTEIN, R. A. Cox AND J. A. HUNT, Biochem. J., 84 (1962) 9 I P . H. ]R. V. ARNSTEIN AND 1R. A. COX, Bioehem. J., 88 (1963) 27P. S. FISCHER, G. P. BRUNS, ]3. A. LowY AND I. M. LONDON, Proc. Natl. Acad. Sci. U.S., 49 (1963) 219. 10 B. I-IARDESTY, R. ARLINGHAUS, J. SHAEFFER AND R. SCHWEET, Cold Spring Harbor Symp. Quant. Biol., 28 (1963) 215. 11 A. P. MATHIAS, R. WILLIAMSON, H. E. HUXLEY AND S. PAGE, J. Mol. Biol., 9 (I964) 154. 12 j. I~RUH, J. C. DREYFUS AND G. SCHAPIRA, Biochim. Biophys. Acta, 87 (1964) 253. 13 j . SHAEFFER, G. FAVELUKES AND R. SCHWEET, Biochim. Biophys. ,4cta, 80 (1964) 247. 14 j . j. ]3ARLOW, A. P. MATH1AS, R. WILLIAMSON AND D. ]3. GAMMACK, Biochem. Biophys. Res. Commun., 13 (1963) 61. 15 j. ]3. LINGREL AND H. ]3ORSOOK, Biochemistry, 2 (1963) 309. 15 M. W. NIRENBERG AND J. H. MATTHAEI, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1588. 1~ R. A. Cox AND H. R. V. ARNSTEIN, Biochem. J., 89 (1963) 574. z p. A. MARKS, C. WILLSON, J. I~RUH AND F. GROS,
2 3 4 5 e 7 s 9
Received September I8th, 1004 Biochim. Biophys. Acta, 91 (1964) 680-683
PN 91037
Presence d'hybrides noturels dons les cellules d'un h~potome oscitique Nous avons signal6 la pr6sence d'un complexe R N A - D N A ayant les caract~res d'un hybride naturel dans la chromatine du noyau de eellules de mammif~res 1. Nous apportons ici des donn6es sur l'isolement et les caract~res des hybrides naturels R N A - D N A , trouv6s clans un h @ a t o m e ascitique. , Nos essais portent sur l'h@atome ascitique de Zajdela obtenu par injection de I ml de liquide d'ascite de la souche par voie intrap~riton6ale ~ des rats Wistar. Au bout de 8 jours et 1. 5 h apr~s une injection intrap~riton~ale de 3oo/~C de H38~PO4 pour IOO g de poids animal, on retire le liquide d'ascite. L'extraction des acides nucl6iques des cellules d'ascite est effectu6e selon KaY ~. La fraction d'acides nucl6iques qui pr~cipite sous forme de fibres est trait6e par le ph6nol z. Le pr6cipit6 fibreux d'acides nucldiques obtenu 5. partir de la phase aqueuse apr~s addition d'un volume d'alcool est lay6, redissous dans o.I M de NaC1 et repr6cipit6 par l'alcool. Le fractionnement des acides nucl6iques sur albumine m6thylde est r6alis6 selon MANDELL ET HERSHEY~ et l'6lution effectn6e par un gradient lin6aire de NaC1 dans le tampon Tris o.oi M (pH 7.1). Pour les digestions ribonuclfiasiques, on utilise Biochim. Biophys..dcla, 91 (1964) 683-686
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.)
G. P.
GEORGIEV
M.I.
LERMAN
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
681
PRELIMINARY NOTES
The cell-free system employed to assay the RNA for messenger activity was basically that of IXTIRENBERG AND MATTHAE116.The RNA to be assayed was added to this system and incubated at 37 ° for 20 rain. The reaction was terminated by the addition of I ml of cold water and 3 ml of 12 % trichloroacetic acid. After standing at 4 ° for at least an hour the precipitate was dissolved in I ml of 0.5 N NaOH and heated at 37 ° for 45 rain. The protein was precipitated by the addition of 4 ml of 12 % trichloroacetic acid, the volume made up to 12 ml using a 7 % solution of this acid and the precipitate collected on a o.45-ff millipore filter. The filters were washed twice with 2 ml of 7 % trichloroacetic acid and glued to the back of aluminum counting planchets. They were dried by standing at room temperature overnight and counted in a Nuclear Chicago gas flow counter with a micromil window. When increasing amounts of reticulocyte ribosomal RNA were added to the E. coli cell-free system there was a corresponding increase in the amount of uniformly
/2\'o
8.00
/ .c_ £
/
o
3000
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/
i6oo
!
E 5.0C
f~f
!
/
=
500 I
/J,
',
?!
~
/ /
/
-lOOOg
8
"~ 2000
,4.oc /
'o,' J
#
..o
< z
~
L 1000
/
<
-500 x /
20C-
ig 0
65 RNA (rag/assay)
1:0
O;
0
~
- ~
,
,
,~ -~:: 40
10 20 30 Fraction number'
Fig. I. Effect of reticulocyte ribosomal RNA concentration on uniformly 14C-labeled amino acid incorporation. The messenger RNA assay system (I.O ml) contained: 1. 4 mg of preincubated $30 protein16; o.5 mg of transfer RNA (both $30 and transfer RNA were from E. coli strain ATCC 8739); o. I 5 # C uniformly ~4C-labeled amino acids (1.38 mC/mg) obtained from New England Nuclear (Chlorella pyrenoidosa algal protein hydrolysate); 0.075 #mole of unlabelled glutamine, cysteine, tryptophan, methionine and asparagine; ioo/*moles Tris-HCI buffer (pH 7.8 at 20°); IO/,moles MgC12; 7 °/,moles KC1; 6/,moles fl-mercaptoethanol; I/,mole ATP; 6/,moles creatine phosphate; o.o3/,mole GTP; 24/,g creatine kinase (EC 2.7.3.2); plus the RNA to be assayed. Messenger RNA activity data are given as counts/min in total protein of the assay system over the control (minus reticulocyte RNA) value of lO25 counts/min. Fig. 2. Sedimentation profile obtained b y sucrose density gradient centrifugation of RNA prepared by phenol extraction. A 6.3-mg sample of ribosomal RNA was layered onto a 28 ml 4-20 % (w/v) linear sucrose gradient in o.o4 M Tris-HCl buffer (pH 7.3). The sucrose solution was previously treated with bentonite (3 mg/ml). The gradient was run at 24 ooo rev./min for 17 h in the Spinco Model L Ultracentrifuge using the SW 25.1 swinging bucket rotor. Fractions were obtained by puncturing the b o t t o m of the tube with a 2o-gauge needle and collecting drops. For assay, 0.25 ml of each fraction was added to the assay system. Messenger RNA activity data ( O - O ) are given as counts/min in total protein of the assay system over the control assay containing 0.25 ml of the appropriate sucrose solution. Absorbancy at 260 m/* ( O - - - O ) .
Biochim. Biophys..4cta, 91 (1964) 680-683
6S2
PRELIMINARY NOTES
~4C-labeled amino acids incorporated into the trichloroacetic acid precipitate. This increase is directly proportional to the amount of RNA added up to about o.5 mg (Fig. i). The incorporation was dependent upon ribosomes and an energy source and was inhibited by puromycin (Table I). TABLE I PROPERTIES
OF
THE
ASSAY
SYSTEM
The complete messenger R N A assay s y s t e m (see legend to Fig. i) was used with and w i t h o u t 0. 5 mg of reticulocyte ribosomal RNA, D a t a are given as c o u n t s / m i n in the total protein of the assay. Activity (counts[minlassay)
Assay conditions
Without RNA
with RNA
iooo ,235 257 265 25 °
.3900 235 298 285 245
Complete s y s t e m precipitated at zero time plus o . o 7 # m o l e puromycin minus ribosomes m i n u s energy* ATP, creatine phosphate, creatine kinase.
To determine if the messenger activity was found in a specific RNA fraction the reticulocyte RNA was fractionated by sucrose density gradient centrifugation and the fractions assayed for messenger activity. As shown in Fig. 2, the messenger activity is not equally distributed among the RNA's but is located in a fraction which migrates just behind the I7-S RNA. To help eliminate the possibility that the active fraction might result from RNA degradation during the phenol isolation, reticulocyte ribosomal RNA was prepared by the guanidine hydrochloridO 7 and lithium chloride 14methods. Both these methods yielded RNA preparations whose messenger RNA activity migrated in an identical position to that found for the messenger RNA activity of the phenol-isolated RNA. Although the method of isolation of the RNA is not critical, the presence of bentonite is and may account for the inability of other workers to fractionate the messenger RNA activity. If bentonite is not present in the isolation, variable activities are obtained. It should be noted that the peak of the messenger RNA activity consistently migrates slightly behind the absorbance peak of the small ribosomal RNA. This unequal distribution suggests an incomplete identity with the small ribosomal RNA. In fact, preliminary experiments show that the small ribosomal RNA peak can be separated into two fractions by methylated albumin chromatography. The demonstration of an RNA fraction which enhances amino acid incorporation into protein in a cell-free system, however, does not prove that one is dealing with a hemoglobin specific messenger RNA. In fact SHAEFFERet a/.lahave presented evidence against the synthesis of hemoglobin in an E . colt system when reticulocyte RNA is added. In contrast KRUH et al. ~ have shown that adding reticulocyte RNA to a reticulocyte cell-free system increases the synthesis of hemoglobin. The question of whether the RNA fraction reported in this communication is specific for hemoglobin must therefore await further studies. Biochim.
Bit)phys. Acla, 9x (1964) 680-683
683
PRELIMINARY NOTES
This investigation was supported by Public Health Service grant GM-Io999 from the National Institute of General Medical Sciences and General Research Support Grants FR-SoI-o518-oI and Soi-FR-o54o8oi.
Department o/ Biological Chemistry, University o/ Cincinnati Collegeo/Medicine, Cincinnati, Ohio (U.S.A.)
J O H N C. DRACH JERRY
B.
LINGREL
Biochem. Biophys. Res. Commun., 8 (1962) 9. D. NATHANS, G. VON EHRENSTEIN, R. MONRO AND V. LIPMANN, Federation Proc., 21 (1962) 127. j . BISHOP, G. FAVELUKES, l°t. SCHWEET AND E. RUSSELL, Nature, 191 (1961) 1365. j. ]3ISHOP AND R. SCHWEET, Biochim. Biophys. Acta, 65 (1962) 553. j . KRUH AND H. ]3ORSOOK, J. Biol. Chem., 220 (1956) 905. H. R. V. ARNSTEIN, R. A. COX AND J. A. HUNT, Nature, 194 (1962) lO42. H. ~_. V. ARNSTEIN, R. A. Cox AND J. A. HUNT, Biochem. J., 84 (1962) 9 I P . H. ]R. V. ARNSTEIN AND 1R. A. COX, Bioehem. J., 88 (1963) 27P. S. FISCHER, G. P. BRUNS, ]3. A. LowY AND I. M. LONDON, Proc. Natl. Acad. Sci. U.S., 49 (1963) 219. 10 B. I-IARDESTY, R. ARLINGHAUS, J. SHAEFFER AND R. SCHWEET, Cold Spring Harbor Symp. Quant. Biol., 28 (1963) 215. 11 A. P. MATHIAS, R. WILLIAMSON, H. E. HUXLEY AND S. PAGE, J. Mol. Biol., 9 (I964) 154. 12 j. I~RUH, J. C. DREYFUS AND G. SCHAPIRA, Biochim. Biophys. Acta, 87 (1964) 253. 13 j . SHAEFFER, G. FAVELUKES AND R. SCHWEET, Biochim. Biophys. ,4cta, 80 (1964) 247. 14 j . j. ]3ARLOW, A. P. MATH1AS, R. WILLIAMSON AND D. ]3. GAMMACK, Biochem. Biophys. Res. Commun., 13 (1963) 61. 15 j. ]3. LINGREL AND H. ]3ORSOOK, Biochemistry, 2 (1963) 309. 15 M. W. NIRENBERG AND J. H. MATTHAEI, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1588. 1~ R. A. Cox AND H. R. V. ARNSTEIN, Biochem. J., 89 (1963) 574. z p. A. MARKS, C. WILLSON, J. I~RUH AND F. GROS,
2 3 4 5 e 7 s 9
Received September I8th, 1004 Biochim. Biophys. Acta, 91 (1964) 680-683
PN 91037
Presence d'hybrides noturels dons les cellules d'un h~potome oscitique Nous avons signal6 la pr6sence d'un complexe R N A - D N A ayant les caract~res d'un hybride naturel dans la chromatine du noyau de eellules de mammif~res 1. Nous apportons ici des donn6es sur l'isolement et les caract~res des hybrides naturels R N A - D N A , trouv6s clans un h @ a t o m e ascitique. , Nos essais portent sur l'h@atome ascitique de Zajdela obtenu par injection de I ml de liquide d'ascite de la souche par voie intrap~riton6ale ~ des rats Wistar. Au bout de 8 jours et 1. 5 h apr~s une injection intrap~riton~ale de 3oo/~C de H38~PO4 pour IOO g de poids animal, on retire le liquide d'ascite. L'extraction des acides nucl6iques des cellules d'ascite est effectu6e selon KaY ~. La fraction d'acides nucl6iques qui pr~cipite sous forme de fibres est trait6e par le ph6nol z. Le pr6cipit6 fibreux d'acides nucldiques obtenu 5. partir de la phase aqueuse apr~s addition d'un volume d'alcool est lay6, redissous dans o.I M de NaC1 et repr6cipit6 par l'alcool. Le fractionnement des acides nucl6iques sur albumine m6thylde est r6alis6 selon MANDELL ET HERSHEY~ et l'6lution effectn6e par un gradient lin6aire de NaC1 dans le tampon Tris o.oi M (pH 7.1). Pour les digestions ribonuclfiasiques, on utilise Biochim. Biophys..dcla, 91 (1964) 683-686