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The effect of nuclear RNA on nuclear protein synthesis
Effect of nuclear 5. 6. 7. 8. 9. 10.
RNA
on nuclear
protein
DAOUST,R. and CANTERO,A., J. Histochem. Cytochem. MCMANUS,J. E. A., Nature (Lond.) 158, 202 (1946). SCHNEYER,L. H. and SCHNEYER,C. A., Am. J. Physiol. SMITHIES,0. Biochem. J. 61, 629 (1955). TODD, A. S. J. Pathof. Bacf. 78, 281 (1959). TREMBLAY,G. J. Histochem. Cyfochem. 11, 202 (1963).
THE
EFFECT
OF NUCLEAR PROTEIN
177
synthesis
7, 139 (1959).
RNA
187, 403 (1956).
ON NUCLEAR
SYNTHESIS
B. B. BISWAS Bose Institute,
Calcutta,
India
Received July 16, 1963
FmM
calf thymus nuclei, a system capable of incorporating nucleoside triphosphates into polynucleotides has been isolated [l] and this was found to be similar to RNA1 polymerase reported from different organisms [5]. It has been observed that nuclear ribosomes were capable of synthesizing protein [4, 71. Ribosomal protein synthesis could be increased by the addition of enzymatically synthesized RNA [8]. It was the purpose of this work to find out whether nuclear ribosomal protein synthesis could be influenced by enzymatically synthesized nuclear RNA. Nuclei from thymus were isolated as described earlier [l]. The nuclei were then homogenized with 5 vol. of 0.1 M Tris buffer containing 0.003 M MgCl, at pH 7.4 and centrifuged at 10,000 xg for 10 min. The pellet was used for nuclear RNA synthesis. The supernatant was further centrifuged at 105,000 x g for 1 hr. The pellet constituted the nuclear ribosomal particle and was dispersedin minimal amount of 0.01 M Tris buffer +0.003 M MgCl, of pH 7.0. From the supernatant pH 5 enzyme was isolated. Nuclear RNA was prepared by incubating the nuclear enzyme (10,000 x g pellet) with 2 ,t&f of eachribonucleosidetriphosphates for 10min. The bulk of the RNA from the enzyme complex was isolated with phenol. 85 per cent of the newly synthesized RNA can be extracted by this method along with the preformed RNA. In order to test the presence of RNA polymerase the ribosomal particles isolated from thymus nuclei were incubated with each labeled nucleoside triphosphate in presenceor absenceof other non-radioactive nucleoside triphosphates. The maximal incorporation of each nucleoside triphosphate is recorded in Table I. It was found that ATP and CTP were actively incorporated whether these were present alone or along with the mixture of other nucleosidetriphosphates but very little activity with GTP or UTP, indicating a possible participation of ATP and CTP polymerases re1 RNA, ribonucleic acid; RNAase, ribonuclease;DNAase, deoxyribonuclease;Tris, tris (bydroxymethyl) aminomethane;RNP, ribonucleoproteinparticle; ATP, CTP, UTP and GTP, the corresponding 5’-triphosphates. 12-
631815
Experimental
Cell Research
32
B. B. Biswas ported from thymus nuclei [3]. The incorporation of ATP was found to be insensitive to the action of pancreatic DNAase or RNAase. These nuclear ribosomal particles seemed to be incapable of synthesizing typical RNA molecules as was found with larger particulate preparation from nuclei [ 11. The effect of nuclear RNA on amino acid incorporation is shown in Table II. Leucine incorporation by the RNP particles required nuclear pH 5 enzyme, ATP and ATP generating system. If any one of them was omitted the incorporation was decreased appreciably. When enzymatically synthesized nuclear RNA was added there was at least a fourfold stimulation in leucine incorporation indicating that nuclear RNA has some effect on protein synthesis by the RNP particles. However, there was at least a two-fold stimulation when particulate endogenous RNA was added to the system. It has been proposed by Brenner ef al. [2] that messenger RNA has a role in cell protein synthesis. The nuclear RNA which reflects the composition of DNA has been shown to act as messenger RNA [8]. The results shown here indicate that the messenger TABLE
I. Incorporation nuclear
of ribonucleoside trisphosphates ribosomal particles.
by
pplcMof nucleosidetriphospateincorporated CTP 320
ATP 230
GTP 5
UTP 5
The incubationmixture containsthe following:RNP particles7-8 mg protein, 0.2 and 0.006 mg DNA/mg protein, 100 @4; Tris buffer (pH 8.0), 5 @4; MgCl,, lO@f;
mg RNA Cysteine, 0.1 @f labeled nucleoside triphosphates in a total vol. of 1 ml; specific activity of *H CTP, WI GTP, 8.2 x 106 counts/min/yM; 12 x lo8 counts/min/@f; I%,, ATP, 1.2 x lo6 counts/min/pM; SH UTP, 4.5 x lo8 counts/min/@f. Incubated for 15 min. The reaction was stopped by addition of 1 ml of 1 M HClO,. TABLE
II.
Incorporation
of
TXeucine
by nuclear
ribosomal
particles.
Sp. ac. of protein counts/min/mg Complete system Complete system - RNP particles Complete system-pH 5 enzyme Complete system+nuclear RNA Complete system+nuclear RNA enzymatically synthesized
(100 (100
,ug) pg)
80 40 35 180 340
The complete system contains RNP particles (7-8 mg protein), nuclear pH 5 enzyme (l-2.5 mg protein), 0.2 mg pyruvate kinase, 10 ,uM sodium phosphoenol pyruvate, 2 PM ATP, 2 @f GTP, 10 PM KCl, 1 ,uM nn-W-leucine of specific activity 3 x 106 counts/min/pM, total volume 2 ml. Incubated for 60 min at 37’. Protein was estimated by Lowry’s method [6]. Experimental
Cell Research
32
Effect
of osmium-fixation
on bacferial
179
flagella
RNA or the nuclear RNA does not only play a role in cytoplasmic protein synthesis but also influences the nuclear protein synthesis. It is to be found whether net protein synthesis can be demonstrated by these particles under similar condition. Thanks are due to Dr. R. Abrams
for his suggestions and criticisms. REFERENCES
1.
2. 3. 4.
5. 6. 7. 8.
BISWAS, B. B. and ABRAMS, FL, Biochim. Biophys. Acta 55, 827 (1962). BRENNER, S., JACOB, F. and MESELSON, M., Nature 190, 576 (1961). EDMONDS, M. and ABRAM?,, R., J. Biol. Chem. 235, 1142 (1960). FRENSTER, J. H., ALLFREY, V. G. and MIRSKY, A. E., Biochim. Biophys. Acta GRUNBERG-MANAGO, &I., Arm. Rev. Biochem. 31, 301 (1962). LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L. and RANDALL, R. J., J. Biol. (1951). WANG, TUNG-YUE, Biochim. Biophys. Acta 51, 180 (1961). WOOD, W. B. and BERG, P., Proc. Natl. Acad. Sci. U.S. 48, 94 (1962).
THE
EFFECT
OF OSMIUM-FIXATION
BACTERIAL B. FORSLIND Department
of Medical
Physics, Received
47,130
(1961).
Chem.
193,265
ON
FLAGELLA and
G. SWANBECK
Karolinska July
Institutet, 16,
Stockholm,
Sweden
1963
MOST
e ltec ron microscopic studies on bacterial flagella have been performed on heavy metal shadowed specimens[2, 6, 7, 8, 91. The flagella appear as long strands with diameters varying between 100-200 A, depending upon the bacterial speciesexamined. The flagella have a length of the order of several microns in intact bacteria but appear considerably shorter when isolated. With refined shadowing techniques some authors have shown a fine structure of the flagellum revealed as thin filaments coiled around each other. These authors therefore regard the flagellum as a structure built as a double or triple helix of filaments [2, 6, 7, 8, 91. Negatively stained pictures of flagella whether treated with ultrasonic vibration, detergents or freezing and thawing do not show the coiled structure mentioned above 151.In the electron micrographs of flagella treated with prolonged ultrasonic vibration one finds small fragments with a diameter of 50 A which have been interpreted as fimbriae, i.e. filamentous appendagesof bacteria not identical with flagella. Material and methods.-The flagella were obtained from a strain of Proteus uulgaris which had been grown on a liquid broth medium in a growth tank of 600 1. These flagella were separated from the bacterial bodies according to the procedure described by Erlander et al. [3]. However, concentrating flagellar suspensionwas performed by
Experimental
Cell Research
32
RNA
on nuclear
protein
DAOUST,R. and CANTERO,A., J. Histochem. Cytochem. MCMANUS,J. E. A., Nature (Lond.) 158, 202 (1946). SCHNEYER,L. H. and SCHNEYER,C. A., Am. J. Physiol. SMITHIES,0. Biochem. J. 61, 629 (1955). TODD, A. S. J. Pathof. Bacf. 78, 281 (1959). TREMBLAY,G. J. Histochem. Cyfochem. 11, 202 (1963).
THE
EFFECT
OF NUCLEAR PROTEIN
177
synthesis
7, 139 (1959).
RNA
187, 403 (1956).
ON NUCLEAR
SYNTHESIS
B. B. BISWAS Bose Institute,
Calcutta,
India
Received July 16, 1963
FmM
calf thymus nuclei, a system capable of incorporating nucleoside triphosphates into polynucleotides has been isolated [l] and this was found to be similar to RNA1 polymerase reported from different organisms [5]. It has been observed that nuclear ribosomes were capable of synthesizing protein [4, 71. Ribosomal protein synthesis could be increased by the addition of enzymatically synthesized RNA [8]. It was the purpose of this work to find out whether nuclear ribosomal protein synthesis could be influenced by enzymatically synthesized nuclear RNA. Nuclei from thymus were isolated as described earlier [l]. The nuclei were then homogenized with 5 vol. of 0.1 M Tris buffer containing 0.003 M MgCl, at pH 7.4 and centrifuged at 10,000 xg for 10 min. The pellet was used for nuclear RNA synthesis. The supernatant was further centrifuged at 105,000 x g for 1 hr. The pellet constituted the nuclear ribosomal particle and was dispersedin minimal amount of 0.01 M Tris buffer +0.003 M MgCl, of pH 7.0. From the supernatant pH 5 enzyme was isolated. Nuclear RNA was prepared by incubating the nuclear enzyme (10,000 x g pellet) with 2 ,t&f of eachribonucleosidetriphosphates for 10min. The bulk of the RNA from the enzyme complex was isolated with phenol. 85 per cent of the newly synthesized RNA can be extracted by this method along with the preformed RNA. In order to test the presence of RNA polymerase the ribosomal particles isolated from thymus nuclei were incubated with each labeled nucleoside triphosphate in presenceor absenceof other non-radioactive nucleoside triphosphates. The maximal incorporation of each nucleoside triphosphate is recorded in Table I. It was found that ATP and CTP were actively incorporated whether these were present alone or along with the mixture of other nucleosidetriphosphates but very little activity with GTP or UTP, indicating a possible participation of ATP and CTP polymerases re1 RNA, ribonucleic acid; RNAase, ribonuclease;DNAase, deoxyribonuclease;Tris, tris (bydroxymethyl) aminomethane;RNP, ribonucleoproteinparticle; ATP, CTP, UTP and GTP, the corresponding 5’-triphosphates. 12-
631815
Experimental
Cell Research
32
B. B. Biswas ported from thymus nuclei [3]. The incorporation of ATP was found to be insensitive to the action of pancreatic DNAase or RNAase. These nuclear ribosomal particles seemed to be incapable of synthesizing typical RNA molecules as was found with larger particulate preparation from nuclei [ 11. The effect of nuclear RNA on amino acid incorporation is shown in Table II. Leucine incorporation by the RNP particles required nuclear pH 5 enzyme, ATP and ATP generating system. If any one of them was omitted the incorporation was decreased appreciably. When enzymatically synthesized nuclear RNA was added there was at least a fourfold stimulation in leucine incorporation indicating that nuclear RNA has some effect on protein synthesis by the RNP particles. However, there was at least a two-fold stimulation when particulate endogenous RNA was added to the system. It has been proposed by Brenner ef al. [2] that messenger RNA has a role in cell protein synthesis. The nuclear RNA which reflects the composition of DNA has been shown to act as messenger RNA [8]. The results shown here indicate that the messenger TABLE
I. Incorporation nuclear
of ribonucleoside trisphosphates ribosomal particles.
by
pplcMof nucleosidetriphospateincorporated CTP 320
ATP 230
GTP 5
UTP 5
The incubationmixture containsthe following:RNP particles7-8 mg protein, 0.2 and 0.006 mg DNA/mg protein, 100 @4; Tris buffer (pH 8.0), 5 @4; MgCl,, lO@f;
mg RNA Cysteine, 0.1 @f labeled nucleoside triphosphates in a total vol. of 1 ml; specific activity of *H CTP, WI GTP, 8.2 x 106 counts/min/yM; 12 x lo8 counts/min/@f; I%,, ATP, 1.2 x lo6 counts/min/pM; SH UTP, 4.5 x lo8 counts/min/@f. Incubated for 15 min. The reaction was stopped by addition of 1 ml of 1 M HClO,. TABLE
II.
Incorporation
of
TXeucine
by nuclear
ribosomal
particles.
Sp. ac. of protein counts/min/mg Complete system Complete system - RNP particles Complete system-pH 5 enzyme Complete system+nuclear RNA Complete system+nuclear RNA enzymatically synthesized
(100 (100
,ug) pg)
80 40 35 180 340
The complete system contains RNP particles (7-8 mg protein), nuclear pH 5 enzyme (l-2.5 mg protein), 0.2 mg pyruvate kinase, 10 ,uM sodium phosphoenol pyruvate, 2 PM ATP, 2 @f GTP, 10 PM KCl, 1 ,uM nn-W-leucine of specific activity 3 x 106 counts/min/pM, total volume 2 ml. Incubated for 60 min at 37’. Protein was estimated by Lowry’s method [6]. Experimental
Cell Research
32
Effect
of osmium-fixation
on bacferial
179
flagella
RNA or the nuclear RNA does not only play a role in cytoplasmic protein synthesis but also influences the nuclear protein synthesis. It is to be found whether net protein synthesis can be demonstrated by these particles under similar condition. Thanks are due to Dr. R. Abrams
for his suggestions and criticisms. REFERENCES
1.
2. 3. 4.
5. 6. 7. 8.
BISWAS, B. B. and ABRAMS, FL, Biochim. Biophys. Acta 55, 827 (1962). BRENNER, S., JACOB, F. and MESELSON, M., Nature 190, 576 (1961). EDMONDS, M. and ABRAM?,, R., J. Biol. Chem. 235, 1142 (1960). FRENSTER, J. H., ALLFREY, V. G. and MIRSKY, A. E., Biochim. Biophys. Acta GRUNBERG-MANAGO, &I., Arm. Rev. Biochem. 31, 301 (1962). LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L. and RANDALL, R. J., J. Biol. (1951). WANG, TUNG-YUE, Biochim. Biophys. Acta 51, 180 (1961). WOOD, W. B. and BERG, P., Proc. Natl. Acad. Sci. U.S. 48, 94 (1962).
THE
EFFECT
OF OSMIUM-FIXATION
BACTERIAL B. FORSLIND Department
of Medical
Physics, Received
47,130
(1961).
Chem.
193,265
ON
FLAGELLA and
G. SWANBECK
Karolinska July
Institutet, 16,
Stockholm,
Sweden
1963
MOST
e ltec ron microscopic studies on bacterial flagella have been performed on heavy metal shadowed specimens[2, 6, 7, 8, 91. The flagella appear as long strands with diameters varying between 100-200 A, depending upon the bacterial speciesexamined. The flagella have a length of the order of several microns in intact bacteria but appear considerably shorter when isolated. With refined shadowing techniques some authors have shown a fine structure of the flagellum revealed as thin filaments coiled around each other. These authors therefore regard the flagellum as a structure built as a double or triple helix of filaments [2, 6, 7, 8, 91. Negatively stained pictures of flagella whether treated with ultrasonic vibration, detergents or freezing and thawing do not show the coiled structure mentioned above 151.In the electron micrographs of flagella treated with prolonged ultrasonic vibration one finds small fragments with a diameter of 50 A which have been interpreted as fimbriae, i.e. filamentous appendagesof bacteria not identical with flagella. Material and methods.-The flagella were obtained from a strain of Proteus uulgaris which had been grown on a liquid broth medium in a growth tank of 600 1. These flagella were separated from the bacterial bodies according to the procedure described by Erlander et al. [3]. However, concentrating flagellar suspensionwas performed by
Experimental
Cell Research
32