Chromatographic Characterization of Amino Acid Transfer and Microsomal Ribonucleic Acids Isolated from Yeast

Fresh bakers’ yeast was obtained from the Red Star Company, Cleveland, Ohio. The procedure for the isolation of the RNA fractions and their designation were those of Crestfield et al. (3) and Davis and Allen (4). Briefly, 150-g batches of finely divided yeast were heated 1 minute at 83-87” and 2 minutes at 80” in a solution of 2% recrystallized Duponol C (sodium lauryl sulfate, U.S.P., du Pont), 4.5% ethanol, and 0.025 M potassium phosphate buffer, pH 6.85. The solution was cooled and centrifuged at 0”. The RNA in the supernatant solution was precipitated and washed with ethanol and then redissolved in water. Sodium chloride was then added to the RNA solution to a final concentration of 1 M. The RNA in the gel which formed was separated by centrifugation and designated RNA A. The RNA in the supernatant fraction was reprecipitated with ethanol and redissolved in a smaller volume of water. Sodium chloride was added to a final concentration of 1 M. The RNA in the precipitate which formed was designated RNA B. The RNA precipitable by ethanol from the final supernatant fraction was called RNA C. After the initial column chromatographic experiment, which established the similarity of the major portions of RNA B and RNA C, they were isolated as a single fraction, RNA B + C, from the supernatant fraction after the separation of RNA A. The isolated RNA A and RNA B -fC fractions were treated by the phenol procedure (5). The fractions A, B, and C, and those isolated by ultracentrifugation were characterized by chromatography on Ecteola (Kutlir Laboratories, Monmouth Junction, New Jersey) ion exchange adsorbent (Column A) as described elsewhere (1,2). RNA I can be eluted from Ecteola adsorbent with a neutral salt solution. A large scale preparation of RNA I was as follows: a column of Ecteola adsorbent 6.5 cm in diameter and 8.5 cm high was prepared

Different RNA fractions present in the cytoplasm of rat liver have been separated by the use of diethylaminoethyl cellulose (Ecteola), an ion exchange adsorbent (1,2). This communication describes the chromatographic characteristics of yeast ribonucleic acid fractions prepared by differential solubility in sodium chloride (3,4) and by ultracentrifugation.

Materials and Methods
Fresh bakers' yeast was obtained from the Red Star Company, Cleveland, Ohio. The procedure for the isolation of the RNA fractions and their designation were those of Crestfield et al. (3) and Davis and Allen (4). Briefly, 150-g batches of finely divided yeast were heated 1 minute at 83-87" and 2 minutes at 80" in a solution of 2% recrystallized Duponol C (sodium lauryl sulfate, U.S.P., du Pont), 4.5% ethanol, and 0.025 M potassium phosphate buffer, pH 6.85. The solution was cooled and centrifuged at 0". The RNA in the supernatant solution was precipitated and washed with ethanol and then redissolved in water. Sodium chloride was then added to the RNA solution to a final concentration of 1 M. The RNA in the gel which formed was separated by centrifugation and designated RNA A. The RNA in the supernatant fraction was reprecipitated with ethanol and redissolved in a smaller volume of water.
Sodium chloride was added to a final concentration of 1 M. The RNA in the precipitate which formed was designated RNA B. The RNA precipitable by ethanol from the final supernatant fraction was called RNA C. After the initial column chromatographic experiment, which established the similarity of the major portions of RNA B and RNA C, they were isolated as a single fraction, RNA B + C, from the supernatant fraction after the separation of RNA A. The isolated RNA A and RNA B -f-C fractions were treated by the phenol procedure (5).
The fractions A, B, and C, and those isolated by ultracentrifugation were characterized by chromatography on Ecteola (Kutlir Laboratories, Monmouth Junction, New Jersey) ion exchange adsorbent (Column A) as described elsewhere (1,2).
RNA I can be eluted from Ecteola adsorbent with a neutral salt solution.
A large scale preparation of RNA I was as follows: a column of Ecteola adsorbent 6.5 cm in diameter and 8. 5  A small fraction of ultraviolet-absorbing material was eluted in tubes 12 to 26 in which the normality of the NaCl was 0.02 to 0.08. The elution of RNA I began in tube 37, where the NaCl concentration was 0.23 N. RNA I was then eluted with 1 N NaCl buffered as above. Approximately 80 mg of RNA were recovered as RNA I. The remaining 70 mg were eluted with 1 N NaOH after all the RNA I had come off.

The supernatant
and microsomal fractions of yeast were isolated by breaking the cells in a Nossal shaker (37.5 g of yeast per 100 ml of solution containing 2.5 X 10e3 M potassium phosphate buffer, pH 7.0, 5 X 10e4 M MgClz shaken at 0" for 15 seconds). The resulting material was centrifuged at 10,000 x g for 15 minutes and then the supernatant fraction was centrifuged at 105,000 x g for 1 hour.
The RNA of the microsomal pellet was extracted initially with the detergent solution (3), precipitated with ethanol, reextracted with 10% NaCl and reprecipitated with ethanol before chromatography (Fig. 2). The microsomal RNA used in the separation reported in Table II was prepared by the phenol method (5).
The methods of measurement of C%leucine incorporation into RNA catalyzed by a rat liver preparation have been described (2). The average sedimentation coefficients (szo,~) were determined by Dr. Herbert Rosenkranz. The RNA fractions were examined in 0.2 N NaCl by the ultraviolet optical system. The electrophoretic analysis on starch was performed as described previously (1). Weight of RNA in milligrams was calculated on the basis of 23.9 optical density units at 260 rnp, pH 7.0, as equivalent to 1 mg. This value was determined from optical density and organic phosphate analyses of two different samples of RNA I. Pentose was estimated by the orcinol method (6) with AMP as a standard.

RESULTS
Data on the distribution of RNA in fractions isolated by differential solubility in NaCl and by ultracentrifugation are presented in Table I. RNA A, insoluble in 1 N N&l, was approximately 80% of the total RNA. The remaining 20% was present in fractions B and C, initially soluble in 1 N NaCl. On separation by ultracentrifugation, 80 % of the RNA recovered was isolated in the microsomal fraction while the remainder was in the supernatant fraction.
Thus the relative proportions recovered as RNA A and microsomal RNA by these two procedures of isolation are comparable.

Crestfield et al. (3) have
reported that 90% of the total RNA in yeast is recovered by the detergent method.
The lower yield of RNA reported in Table I for the fractions recovered by ultracentrifugation is assumed to be primarily due to lack of disruption of all yeast cells by the Nossal shaker.
Chromatographic analysis of the fractions obtained by differential solubility in NaCl and by ultracentrifugation was performed by the use of Ecteola ion exchange adsorbent.
Elution patterns of the RNA fractions obtained by fractionation with NaCl are presented in Figs. 1 and 2. The major portions of both RNA B and RNA C (Fig. 1) were eluted with the NaCl gradient, i.e. 70% of RNA B and 48% of RNA C. In this preparation a significant proportion of RNA C (38 %) was eluted before tube 12. In subsequent elution patterns of large amounts of the combined RNA B + C, the proportion of this material was considerably less. RNA B, 30%, and RNA C, 14%, were found in the material eluted with alkali.
The RNA fraction eluted between tubes 20 and 44 behaved chromatographically exactly like an RNA fraction obtained from the high speed supernatant material of rat liver which was shown to act as an amino acid acceptor (1,2). This rat liver RNA has been designated RNA I. Therefore, a large scale chromatographic isolation procedure of the analogous RNA I from the combined yeast RNA B + C fractions was developed as described in the methods section.
The preparation after chromatography contained less contaminating material. Rechromatography of yeast RNA I on a small Ecteola column resulted in recovery of 68% as RNA I, 15% as RNA eluted with alkali, and 17 To as RNA eluted before RNA I. On rechromatography of the 68% RNA A, the material initially insoluble in 1 N NaCl, produced the elution pattern shown in Fig. 2. Very little material was eluted with the NaCl gradient, whereas almost all of the material was eluted with alkali.
On rechromatography of the RNA    The results of the chromatographic characterization of the RNA fractions, obtained by ultracentrifugation of the material from yeast cells broken in the Nossal shaker, are presented in Table II. Of the RNA in the supernatant material after centrifugation at 105,000 x g for 1 hour 69% was eluted with NaCl at a position characteristic of RNA I, and the remainder was eluted with alkali.
The RNA, isolated from the microsomal pellet which was shown to consist of particles with an average sedimentation coefficient (szo,~) of 84, contained no RNA I as indicated both in Table II and in Fig. 2. When this RNA fraction, eluted with 0.1 N NHdOH, 1 N NaCl, was dialyzed and rechromatographed, 88% was recovered at the same point and none was recovered as RNA I. The RNA from the supernatant material, obtained after centrifugation at 10,000 X g for 15 Yeast Ribonucleic Acids Vol. 235, No. 7 minutes, was fractionated on the Ecteola column; 14% of the RNA was isolated as RNA I. This value for the combined fractions coincides closely with the percentage calculated from the data of the separate supernatant and microsomal fractions (Tables I and II).
Known amounts of RNA I and microsomal RNA, isolated by chromatography, could be mixed, rechromatographed and recovered with a 24% loss of RNA I and a 5% loss of microsomal RNA.
The chromatographic artifact described above may account for approximately 15% of the RNA I which was lost.
The effect of the RNA fractions on the incorporation of U4leucine incubated in the presence of a rat liver enzyme preparation for 15 minutes is indicated in Fig. 3. The data demonstrate that fractions B and C were active while fraction A was not. The lower activity of RNA C may be due to the presence of the material noted in the elution pattern before RNA I. The incorporation of U4-leucine as a function of time in the presence of 1.44 mg of RNA B + C is indicated in Fig. 4. The stimulation of C14-leucine incorporation by RNA I isolated from several preparations of RNA B + C was also studied. The results, presented in Table III, indicate that the column purification did not seem to increase the activity significantly.
The reason for the higher activity of the preparation used for the experiments in Figs. 3 and 4 compared to the preparations tested in Table  III is not apparent.
In order to establish further the validity of the chromatographic separation, RNA I and RNA A were mixed, chromatographed, and reisolated. The ability of these fractions to stimulate Cr4-leucine incorporation was compared with the starting material.
The reisolated RNA I retained 58 '% of its activity whereas the minimal activity in the RNA A was not altered significantly.
In one experiment, RNA B + C was incubated with C14-leucine and the rat liver preparation, reextracted with phenol, and chromatographed.
Each fraction was counted by a scintillation counter. The radioactivity was found throughout the RNA I fraction, indicating that the column did not resolve an RNA specific for leucine.
There was no activity noted in the ultraviolet-absorbing material eluted before RNA I. RNA I and RNA A were examined in the ultracentrifuge by Dr. Herbert Rosenkranz.
As indicated in Table IV, the former had an average sedimentation coefficient (szo,,J of 3.2 whereas the latter had a value of 4.5. The relative mobility of these RNA fractions studied by starch electrophoresis is indicated in Table IV and Fig. 5. The RNA I migrated slightly faster. Analysis of RNA I for phosphate, pentose, and protein is reported in Table IV. The value for protein is corrected for the reaction of guanine with the reagents and represents less than 1 y0 of the weight of the RNA plus protein.
The extinction coefficient of two different samples of RNA I was 7800 and 8100 per mmole of organic phosphate; the average, 7,950, is presented in Table IV. DISCUSSION Amino acid transfer RNA and microsomal RNA present in yeast can be separated by either differential solubility in 1 N NaCl or by ultracentrifugation.
The fraction of RNA containing the amino acid transfer RNA isolated by either method comprises 15 to 20% of the total RNA isolated.
This RNA is eluted from Ecteola adsorbent with NaCl at exactly the same molarity as a fraction of amino acid transfer RNA of rat liver (1, 2) and its behavior on rechromatography is similar. The average sedimentation coefficient (szO,,,,) of the yeast amino acid transfer RNA is 3.2. It is assumed that RNA molecules able to transfer amino acids other than leucine are present in this fraction, because of the association of the various transfer RNA species during preparation by other methods, and the difficulties encountered by several investigators in achieving only partial separation (9,10). Contamination by small amounts of nontransfer RNA cannot be ruled out.
Microsomal RNA represents approximately 80% of the RNA isolated both by the detergent procedure (3,4) and by ultracentrifugation.
This material can be eluted from Ecteola adsorbent at pH values above 9.5. The average sedimentation coefficients (s%,~) of this material is 4.5. Microsomal RNA migrates slower than amino acid transfer RNA on starch electrophoresis.
The behavior of yeast microsomal RNA on Ecteola adsorbent and on electrophoresis is similar in some respects to the postmicrosomal RNA isolated from rat liver (1). However, it differs in that on rechromatography it does not yield significant amounts of the RNA which can be eluted with NaCl, and it does not stimulate amino acid incorporation.
The suggestion has been made that amino acid transfer RNA might be bound in an alkali labile linkage in the postmicrosomal fraction (2). The incubation of yeast microsomal RNA in 0.1 N NH40H at 37" for as long as 60 minutes did not liberate any material with chromatographic characteristics of amino acid transfer RNA. SUMMARY 1. Yeast ribonucleic acid (RNA) fractions obtained by differential solubility in 1 N NaCl and by ultracentrifugation have been examined by chromatography on diethylaminoethyl cellulose (Ecteola) adsorbent.
2. The fraction of yeast RNA which contains amino acid transfer RNA is 15 to 20% of the total RNA. It can be isolated by ultracentrifugation in the high speed supernatant fraction, is soluble in 1 N NaCl, can be eluted partially from Ecteola adsorbent with neutral NaCl solution, and stimulates the incorporation of C4-leucine. 3. The fraction which contains microsomal RNA is 80 to 85% of the total RNA. It can be isolated by ultracentrifugation in the microsomal fraction, is insoluble in 1 N NaCl, can be eluted from Ecteola adsorbent at a pH above 9.5, and does not stimulate CWeucine incorporation.