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Countercurrent distribution of yeast ribonucleic acid “core”
628
BIOCHIMICA ET BIOPHYSICA ACTA
SC 7106
Countercurrent distribution of yeost ribonucleic ocid "core" Fractionation of amino acid-acceptor RNA's by countercurrent distribution have been reported ~-e. We wish to describe here an application of this technique for the fractionation of yeast R N A "core" isolated after pancreatic RNAase digestion of yeast RNA. Also described is the ability of various "core" fractions to enhance the production of streptolysin-S by resting cells of Streptococcus pyogenes (Group A-flhemolytic). The yeast R N A "core" was purchased from Worthington Biochemical Co. (Freehold, N.J., U.S.A.) and had been prepared according to the procedure of HILMOE7. The solvent system, which is a modification of KIRBY'Ss solvent system, was prepared as follows: 12oo g of (NH4)2SO 4 was dissolved in approx. 3000 ml of distilled water and 40 ml of glacial acetic acid was added to it. The solution was made up to 4000 ml (pH 3.25). To this solution was added 16o ml of formamide and 16oo ml 2-ethoxyethanol, and the contents were shaken vigorously. The solvent system was allowed to equilibrate overnight prior to use. Approx. 35o mg of yeast R N A "core" were subjected to 225-transfer distribution. At the end of the distribution, "core" fractions were isolated as the pooled contents of six tubes each. The procedures for performing countercurrent distribution and the isolation of fractions have been described 9. The results are shown in Fig. I.
/\\
75
21.0
o~
.co 5o 10.5 _o
3 3
v >. >
o, 2,5 E
"5
<
50
I00 Tube No.
t50
200
Fig. i. 225-transfer countercurrent distribution of 35 ° m g y e a s t R N A " c o r e " . Q - O , m g R N A " c o r e " per fraction; × - × , e n h a n c e m e n t of streptolysin-S production activity.
The "core" fractions were analyzed for their nucleotide compositions using alkaline hydrolysis and paper chromatography z°, and for their ability to enhance the production of streptolysin-S, using an assay procedure which was a modification of the one described by TANAKA et al. 1~. Freshly withdrawn human red blood cells were Biochim.
Biophys. Acta, 76 (1963) 628-63 °
629
SHORT COMMUNICATIONS
used in these determinations. The nucleotide compositions of various fractions are shown in Table I, whereas the streptolysin-S production activity is represented in Fig. I. TABLE I NUCLEOTIDE CONTENTS OF VARIOUS R N A "CORE" FRACTIONS ISOLATED FROM 225-TRANSFER COUNTERCURRENT DISTRIBUTION OF YEAST R N A "CORE" The figures represented in this table are averages of three determinations. Ap = adenylic acid; Gp = guanylic acid; Cp = cytidylic acid; U p = uridylic acid. Fraction
Tube No.
Ap (%)
C-p (%)
Cp (%)
Up (%)
I 3 5 7 9 II 13 15 17 19
1-6 I3-I8 25-3 ° 37-42 55 ~5o 75-80 95-1oo 115-12o 135-14o 155-16o
13.9 13.9 15.1 12.8 16.7 18. 3 25.8 33.7 39.o 50.3
68.0 74.9 64.9 53.1 48.0 44.6 43.6 41-5 39.4 25.1
8.9 3.3 13.9 21. 5 22.9 23. 7 19.9 13.3 8.o 8.8
9.I 7.9 9.3 11. 7 12. 4 13. 4 lO.9 11.9 13.6 15.8
The guanylic acid contents of the fractions appear to decrease with the increase m partition coefficient whereas the reverse trend is observed in the case of adenylic acid. Cytidylic acid content is lower in the fractions of lower and higher partition coefficient, whereas it is high in the intermediate fractions. The uridylic acid contents appear to vary very little with the partition coefficient. The ability to enhance the streptolysin-S production appears to be associated mainly with the first peak, which has a high guanylic acid content. This is in agreement with the results obtained by ISlalKURATM. The peak fraction in Peak I,contained about 3200 hemolytic units. On the basis of absorption at 260 m# the peak fraction was about 2.5 times as active as the starting material. The results presented here show that countercurrent distribution .technique may be useful in fractionation of oligonucleotides. Preliminary experiments in which the material obtained from Peak I in countercurrent distribution of RNA "core" was subjected to further fractionation by DEAE-cellulose-urea column chromatographyTM, show that this fraction consisted of penta, hexa, hepta, octa, nona ancl deca nucleotides. This indicates that the fractionation obtained by countercurrent distribution is dependent on either the variafIon in nucleotide sequence or the variation in nucleotide composition or both. It is also possible that the variation in nucleotide chain length may contribute in the separation in this solvent system. Experiments are in progress to fractionate the oligonucleotides of the same chain length (obtained by chromatography on DEAE-cellulose-urea column) by countercurrent distribution. This material is taken from a thesis submitted by G. J. M. to Georgetown University, Washington, D.C. (U.S.A.) in partial fulfilment of the requirements for the, degree of Doctor of Philosophy.
Division o/ Medicine and Division o[ Biochemistry, Walter Reed Army Institute o/ Research, Walter Reed Army Medical Center, Washington, D.C. (U.S.A.)
GERALDJ . MCCORMICK B . P . DOCTOR
Biochim. Biophys. A a a , 76 (1963) 628-630
630 I 2 z i b 6 7 s 9 x0 11 lZ 13
SHORT COMMUNICATIONS
1~. W. HOLLRY AND S. H. MERRILL, J. Am. Chem. Sot., 81 (1959) 753]3. P. DOCTOR, J. APGAR AND R. W. HOLLRY, J. Biol. Chem., 236 (I96I) 1117. j . APGAR, R. W. HOLLEY AND S. H. MERRILL, J. Biol. Chem., 237 (1962) 796. ]3. P. DOCTOR AND C. M. CONNRLLY, Biochem. Biophys. Res. Commun., 6 (I96i) 2Ol. I-I. fT. ZACHAU, M. TADA, W. ]3. LAWSON A1KD M. SCHWEIGER, Biochim. Biophys. Acta, 53 (1961) 221. ]3. WRISBLUM, S. ]JENZER AND R. W. HOLLEY, Proc. Natl. Acad. Sci. U.S:, 48 (I962) 1449. R. J. HILMOE, J. Biol. Chem., 235 (196o) 2117. •. S. KIRBY, Biochim. Biophys. Acta, 41 (196o) 338. ]3. p. DOCTOR, C. M. COI~NRLLV, G. W. RUSHIZKY AND H. A. SOBER, J. Biol. Chem. (1963), in the press. G. W. lZOSHIZKY AND H. A. SOBER, J. Biol. Chem., 237 (1962) 834. I~. TANAKA, S. MAEKAWA, T. HAYASHI AND Y. KUROIWA, J. Biochem. Tokyo, 43 (1956) 827. H. ISHIKURA, Biochim. Biophys. Acta, 51 (1961) 189. D. ]JELL, t{.. V. TOMLINSON AND G-. M. TRNER, Biochem. Biophys. Res. Commun., IO (1963) 304 .
Received July Ist, 1963 Biochim. Biophys. Acta, 76 (1963) 628-63o
SC 7107
Further evidence for the existence of a specific ribonucleic acid in liver ribosomes
Our previous experiments1, 2 showed that sonication of the ribosomes of rat liver resulted in a decrease in their incorporating activity with the concomitant release of a small amount of RNA possessing high metabolic activity. In an attempt to provide further evidence for the existence of such a specific RNA in liver ribosomes and to gain more information on its properties and role in protein biosynthesis, the following experiments were undertaken. Liver ribosomes, prepared by the method described in our previous report 1, were obtained from rat liver 30 min after the injection of [14C]orotic acid. They were purified further by ultracentrifugation using a sucrose gradient and were then freed from Mg2+ by dialysis against Mg2+-free medium or by addition of EDTA (5 mM excess over the Mg2+ in the suspension medium*). The pattern of the densitygradient centrifugation of ribosomes after such treatments showed the release of RNA with a high specific activity (about 12-14 times higher than that of the total ribosomal RNA) in the 6- to I5-S region, while that of the original ribosomes showed high specific activity in the region heavier than 135 S (Fig. I). The finding that sonic treatment also released such an RNA in t h e same region, with almost the same specific activity (Fig. Ib), may confirm our previous assumption that sonication releases the specific RMA from ribosomes. The sedimentation diagram of ribosomes at the later stage (2-6 h) of [14C]orotic acid injection, gradually lost such a characteristic pattern in both the original and the treated ribosomes, and that at 15 h after injection showed a radioactivity curve which was coincident with the absorption curve at 260 m/, even in the case of EDTA-treated ribosomes. After liver ribosomes had incompletely lost the incorporating activity of [x4C]leucine into protein by dialysis against MgZ+-free medium at o °, redialysis against 5 mM Mgz+ medium overnight restored the activity partially (Table I). In one of * As a suspending m e d i u m f o r liver ribosomes, m e d i u m A'(5 mM MgC1 v 5 ° mM KCt, i o mM KHCOs, 25 ° m M sucrose and 5 ° mM Tris-HC1 (pH 7.6.)) was used 1.
Biochim. B~ophys. Acta, 76 (1963) 630-632
BIOCHIMICA ET BIOPHYSICA ACTA
SC 7106
Countercurrent distribution of yeost ribonucleic ocid "core" Fractionation of amino acid-acceptor RNA's by countercurrent distribution have been reported ~-e. We wish to describe here an application of this technique for the fractionation of yeast R N A "core" isolated after pancreatic RNAase digestion of yeast RNA. Also described is the ability of various "core" fractions to enhance the production of streptolysin-S by resting cells of Streptococcus pyogenes (Group A-flhemolytic). The yeast R N A "core" was purchased from Worthington Biochemical Co. (Freehold, N.J., U.S.A.) and had been prepared according to the procedure of HILMOE7. The solvent system, which is a modification of KIRBY'Ss solvent system, was prepared as follows: 12oo g of (NH4)2SO 4 was dissolved in approx. 3000 ml of distilled water and 40 ml of glacial acetic acid was added to it. The solution was made up to 4000 ml (pH 3.25). To this solution was added 16o ml of formamide and 16oo ml 2-ethoxyethanol, and the contents were shaken vigorously. The solvent system was allowed to equilibrate overnight prior to use. Approx. 35o mg of yeast R N A "core" were subjected to 225-transfer distribution. At the end of the distribution, "core" fractions were isolated as the pooled contents of six tubes each. The procedures for performing countercurrent distribution and the isolation of fractions have been described 9. The results are shown in Fig. I.
/\\
75
21.0
o~
.co 5o 10.5 _o
3 3
v >. >
o, 2,5 E
"5
<
50
I00 Tube No.
t50
200
Fig. i. 225-transfer countercurrent distribution of 35 ° m g y e a s t R N A " c o r e " . Q - O , m g R N A " c o r e " per fraction; × - × , e n h a n c e m e n t of streptolysin-S production activity.
The "core" fractions were analyzed for their nucleotide compositions using alkaline hydrolysis and paper chromatography z°, and for their ability to enhance the production of streptolysin-S, using an assay procedure which was a modification of the one described by TANAKA et al. 1~. Freshly withdrawn human red blood cells were Biochim.
Biophys. Acta, 76 (1963) 628-63 °
629
SHORT COMMUNICATIONS
used in these determinations. The nucleotide compositions of various fractions are shown in Table I, whereas the streptolysin-S production activity is represented in Fig. I. TABLE I NUCLEOTIDE CONTENTS OF VARIOUS R N A "CORE" FRACTIONS ISOLATED FROM 225-TRANSFER COUNTERCURRENT DISTRIBUTION OF YEAST R N A "CORE" The figures represented in this table are averages of three determinations. Ap = adenylic acid; Gp = guanylic acid; Cp = cytidylic acid; U p = uridylic acid. Fraction
Tube No.
Ap (%)
C-p (%)
Cp (%)
Up (%)
I 3 5 7 9 II 13 15 17 19
1-6 I3-I8 25-3 ° 37-42 55 ~5o 75-80 95-1oo 115-12o 135-14o 155-16o
13.9 13.9 15.1 12.8 16.7 18. 3 25.8 33.7 39.o 50.3
68.0 74.9 64.9 53.1 48.0 44.6 43.6 41-5 39.4 25.1
8.9 3.3 13.9 21. 5 22.9 23. 7 19.9 13.3 8.o 8.8
9.I 7.9 9.3 11. 7 12. 4 13. 4 lO.9 11.9 13.6 15.8
The guanylic acid contents of the fractions appear to decrease with the increase m partition coefficient whereas the reverse trend is observed in the case of adenylic acid. Cytidylic acid content is lower in the fractions of lower and higher partition coefficient, whereas it is high in the intermediate fractions. The uridylic acid contents appear to vary very little with the partition coefficient. The ability to enhance the streptolysin-S production appears to be associated mainly with the first peak, which has a high guanylic acid content. This is in agreement with the results obtained by ISlalKURATM. The peak fraction in Peak I,contained about 3200 hemolytic units. On the basis of absorption at 260 m# the peak fraction was about 2.5 times as active as the starting material. The results presented here show that countercurrent distribution .technique may be useful in fractionation of oligonucleotides. Preliminary experiments in which the material obtained from Peak I in countercurrent distribution of RNA "core" was subjected to further fractionation by DEAE-cellulose-urea column chromatographyTM, show that this fraction consisted of penta, hexa, hepta, octa, nona ancl deca nucleotides. This indicates that the fractionation obtained by countercurrent distribution is dependent on either the variafIon in nucleotide sequence or the variation in nucleotide composition or both. It is also possible that the variation in nucleotide chain length may contribute in the separation in this solvent system. Experiments are in progress to fractionate the oligonucleotides of the same chain length (obtained by chromatography on DEAE-cellulose-urea column) by countercurrent distribution. This material is taken from a thesis submitted by G. J. M. to Georgetown University, Washington, D.C. (U.S.A.) in partial fulfilment of the requirements for the, degree of Doctor of Philosophy.
Division o/ Medicine and Division o[ Biochemistry, Walter Reed Army Institute o/ Research, Walter Reed Army Medical Center, Washington, D.C. (U.S.A.)
GERALDJ . MCCORMICK B . P . DOCTOR
Biochim. Biophys. A a a , 76 (1963) 628-630
630 I 2 z i b 6 7 s 9 x0 11 lZ 13
SHORT COMMUNICATIONS
1~. W. HOLLRY AND S. H. MERRILL, J. Am. Chem. Sot., 81 (1959) 753]3. P. DOCTOR, J. APGAR AND R. W. HOLLRY, J. Biol. Chem., 236 (I96I) 1117. j . APGAR, R. W. HOLLEY AND S. H. MERRILL, J. Biol. Chem., 237 (1962) 796. ]3. P. DOCTOR AND C. M. CONNRLLY, Biochem. Biophys. Res. Commun., 6 (I96i) 2Ol. I-I. fT. ZACHAU, M. TADA, W. ]3. LAWSON A1KD M. SCHWEIGER, Biochim. Biophys. Acta, 53 (1961) 221. ]3. WRISBLUM, S. ]JENZER AND R. W. HOLLEY, Proc. Natl. Acad. Sci. U.S:, 48 (I962) 1449. R. J. HILMOE, J. Biol. Chem., 235 (196o) 2117. •. S. KIRBY, Biochim. Biophys. Acta, 41 (196o) 338. ]3. p. DOCTOR, C. M. COI~NRLLV, G. W. RUSHIZKY AND H. A. SOBER, J. Biol. Chem. (1963), in the press. G. W. lZOSHIZKY AND H. A. SOBER, J. Biol. Chem., 237 (1962) 834. I~. TANAKA, S. MAEKAWA, T. HAYASHI AND Y. KUROIWA, J. Biochem. Tokyo, 43 (1956) 827. H. ISHIKURA, Biochim. Biophys. Acta, 51 (1961) 189. D. ]JELL, t{.. V. TOMLINSON AND G-. M. TRNER, Biochem. Biophys. Res. Commun., IO (1963) 304 .
Received July Ist, 1963 Biochim. Biophys. Acta, 76 (1963) 628-63o
SC 7107
Further evidence for the existence of a specific ribonucleic acid in liver ribosomes
Our previous experiments1, 2 showed that sonication of the ribosomes of rat liver resulted in a decrease in their incorporating activity with the concomitant release of a small amount of RNA possessing high metabolic activity. In an attempt to provide further evidence for the existence of such a specific RNA in liver ribosomes and to gain more information on its properties and role in protein biosynthesis, the following experiments were undertaken. Liver ribosomes, prepared by the method described in our previous report 1, were obtained from rat liver 30 min after the injection of [14C]orotic acid. They were purified further by ultracentrifugation using a sucrose gradient and were then freed from Mg2+ by dialysis against Mg2+-free medium or by addition of EDTA (5 mM excess over the Mg2+ in the suspension medium*). The pattern of the densitygradient centrifugation of ribosomes after such treatments showed the release of RNA with a high specific activity (about 12-14 times higher than that of the total ribosomal RNA) in the 6- to I5-S region, while that of the original ribosomes showed high specific activity in the region heavier than 135 S (Fig. I). The finding that sonic treatment also released such an RNA in t h e same region, with almost the same specific activity (Fig. Ib), may confirm our previous assumption that sonication releases the specific RMA from ribosomes. The sedimentation diagram of ribosomes at the later stage (2-6 h) of [14C]orotic acid injection, gradually lost such a characteristic pattern in both the original and the treated ribosomes, and that at 15 h after injection showed a radioactivity curve which was coincident with the absorption curve at 260 m/, even in the case of EDTA-treated ribosomes. After liver ribosomes had incompletely lost the incorporating activity of [x4C]leucine into protein by dialysis against MgZ+-free medium at o °, redialysis against 5 mM Mgz+ medium overnight restored the activity partially (Table I). In one of * As a suspending m e d i u m f o r liver ribosomes, m e d i u m A'(5 mM MgC1 v 5 ° mM KCt, i o mM KHCOs, 25 ° m M sucrose and 5 ° mM Tris-HC1 (pH 7.6.)) was used 1.
Biochim. B~ophys. Acta, 76 (1963) 630-632