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Thin-layer electrophoresis of ribonucleotides
ANALYTICAL.
BIOCHEMISTRY
Thin-Layer
28, 313-317
Electrophoresis JOAO
Division
of
(1969)
of
Ribonucleotides
P. MONJARDINO
G%emistry and Bioehemistr~, Imperial Cancer Reseamh Lincoln’s Inn Fields, London WC??, England Received August
Fund,
21, 1968
Mild alkaline hydrolysis of RNA and subsequent electrophoresis of the resulting mixture of 2’,3’-nucleotides is now a standard technique in nucleic acid base composition analysis. The extension of its use to characterize RNA labeled with P32 under various conditions has made it an invaluable tool for the study of RNA metabolism of the cell. The method reported here makes possible the completion of a satisfactory electrophoretic separation of ribonucleotides (20-40 pg) on thin layers of cellulose in 90 min at 4OC using only conventional equipment. Base ratio determinations were simple and reproducible. MATERIALS
AND
METHODS
The samples of RNA used included yeast RNA (Sigma type xl), pig pituitary total RNA extracted by a modification of the hot phenol method described by Cline (1)) and nuclear and cytoplasmic pig pituitary RNA’s prepared using the same extraction procedure after isolation of the two fractions according to Hiatt (2) or Roberts et al. (3). Hydrolysis was carried out in 0.3 N KOH (1 mg/ml) at 37OC for 18 hr. The pH of the hydrolyzate was then adjusted to 7.0 by addition of perchloric acid and the precipitate of potassium perchlorate was spun down. The clear supernatant was then transferred into another tube and concentrated in a rotary evaporator before being applied to the plates. The glass plates used (5 X 20 cm) were washed as described previously (4) before being coated with the absorbent. The cellulose (MN 300, Macherey, Nagel & Co.) was washed according to De Fillipes (5) and the plates were coated 500 mp thick using a Dessaga spreader, dried in an over at 100°C, and stored in a 313
314
J. P. MONJARDINO
desiccator. The buffer used was 0.02 M citrate buffer (pH.3.5) freshly prepared prior to each run by diluting 50 times a stock solution made up by the addition of 3 vol 1 M citric acid and 1 vol 1 M trisodium citrate. The sample was applied to the plate 6 cm from one end as a band 1.0-1.5 cm wide. The plate was then immersed in a jar containing the buffer up to 2 or 3 cm below the spotted band and quickly placed in a Shandon electrophoresis tank in such a way that the origin was on the side of the negative electrode; 3MM Whatman wicks which had been dipped in buffer and folded in half were then applied to both ends and the current switched on as soon as the sample zone was wetted by convergence of both buffer fronts. A voltage of 650 V was usually applied (giving a voltage gradient of approximately 50 v/cm) and the current was about 3-5 mA. The separation was carried out at 4OC and was completed in 90 min. The plates were then dried under an infrared lamp and the bands located and penciled round under ultraviolet light (254 rnp) . For determination of base composition the bands and a blank (an area of cellulose from either side of the sample “pathway” of roughly the same size as the bands) were scraped off with a razor blade, transferred to polystyrene tubes containing 0.5 ml of 0.1 N HCl, and eluted overnight. The absorbent was centrifuged and the supernatant transferred to microcuvets for measurement of optical density at the appropriate wavelengths (CMP 279 rnp; AMP 257 rnp; GMP 257 mp; UMP 260 mp; Uvispek spectrophotometer). Conversion to pg nucleotide used millimolar extinction coefficients determined from commercial preparations (Sigma) of the corresponding 5’-nucleotides dissolved in 0.1 N HCl (AMP 15.5; CMP 13.1; GMP 11.6; UMP 10.7). Base composition of the same RNA samples was also determined by the thin-layer chromatography (TLC) method previously reported (4). RESULTS
AND
DISCUSSION
A complete separation of the hydrolyzate mixture into four bands was achieved after each run (Fig. 1). The bands were clearly seen under UV light and were identified by their ultraviolet absorption spectra (Fig. 2) after overnight elution in 0.1 N HCl; they were seen to correspond (starting from the origin) to 2’,3’-CMP, 2’,3’AMP, 2’,3’-GMP, and 2’,3’-UMP, respectively (Fig. 1). The GMP band was sometimes split in two, an observation also reported by Davidson and Smellie (6) and probably due to separation of the 2’ and 3’ isomers.
TLE
OF
Q
(+) 1 () 0 (a)
315
RIBONUCLEOTIDES
(b)
0
(cl
(-)
Cd)
FIG. 1. Thin-layer electrophoresis of 0.3 N KOH hydrolyzate of pig pituitary cytoplasmic ribonucleic acid. The bands starting from the origin are: (a) CMP, (b) AMP, (c) GMP, (d) UMP.
Recoveries of accurately measured amounts of commercial standards (5’-nucleotides from Sigma) were found to be approximately 90%. Base ratio determinations performed on cytoplasmic and nuclear pituitary pig RNA as well as on commercial yeast RNA samples were easy to carry out and reproducible (Table 1). There is also satisfactory agreement with the base compositions obtained by means of the TLC method previously reported (4) (Table 1). The differences found for nuclear RNA are probably explained by the different preparative methods used. The present system shows several advantages when compared with the established electrophoretic separation on paper. It allows for much smaller amounts of RNA to be analyzed-20-40 pg as opposed to 200-800 pg (7) -and the
0.7.
:
0.6 _ 05s
r:.a 0
0.3. \, .._.*
02.
.d
‘.
0 I-
*. ... ... *\ ‘.._ *\ ‘.__‘. -..:.
\
\.
.>.. .._ e:?:.;:.JL.., \
0
220
230
240
260
270
Wavelength,
ny
250
280
290
300
FIG. 2. UV spectra of nucleic acid components separated by TLE from 0.3 N KOH hydrolyzate of pig pituitary RNA: -.-. band (a), CMP (as in Fig. 1);. . . . band (b),AMP; -band (c), GMP; ---band (d), UMP.
316
J. P. MONJARDINO
Base Compositions
TABLE 1 of Pig Pituitary and Yeast RNA’s by TLCd and TLE* Methods (molar percentages)
source
f&Z!
Pig pituitary cytoplasm
Pig pituitary nuclei
G
A
C
U
Method
4
34.1 zt.41
17.2 zk.60
27.8 b.64
20.9 zk.75
TLCd
5
34.1 ~1~1.26
17.6 f.49
26.4 1.71
21.9 f1.43
TLEo
5
34.4 *1.03
18.8 zt1.23
26.0 dz.8
20.9 f.53
TLC’
17.5 f1.21
24.0 1.59
21.6 zk.77
TLE-
30.1 fl.1
25.3 fl.O
20.5 zk1.2
24.1 Al.2
TLCd
31.9 1.82
23.8 Al.1
19.5 fl.33
24.8 fl.1
TLEa
4 Yeast
14
36.9 ht.61
’
0 Present method. b Nuclear fraction prepared according to (2). = Nuclear fraction prepared according to (3). a Previously published (4).
separation is completed in 90 mm instead of the 18 hr for the paper system. Using the latter, the length of the run can be shortened by increasing the voltage gradient but this adds to the technical difficulty of counteracting the excess of heat being generated and calls for special cooling devices which make the whole apparatus cumbersome and expensive. Cur system, on the contrary, allows for a very rapid separation without any special cooling devices being required. SUMMARY
Electrophoresis on thin layers of cellulose of an alkaline hydrolyzate of RNA provides an easy and reproducible method for base ratio determinations. Separation of a nucleotide mixture (20-40 pg) is completed in 90 min at 4OC and requires only conventional equipment. ACKNOWLEDGMENT I wish to thank
Mrs. J. Harman
for her valuable
technical
assistance.
TLE OF RIBONUCLEOTIDES
317
REFERENCES 1. CLINE, M. J., J. Lab. Clin. Med. 68, 33 (1966). 2. HIATT, H. H., J. Mol. Biol. 5, 217 (1962). 3. ROBERTS, W. K., NEWMAN, J. F. E., AND RUECKERT, R. R., J. Mol. Biol. 15, 92 (1966). 4. MONJARDINO, J. P., Anal. B&hem. 21, 308 (1967). 5. DE FILLIPES, F. M., Science 144, 1350 (1964). 6. DAVIDSON, J. N., AND SMELLIE, R. M. S., Biochem. J. 52, 699 (1952). ‘7. SMITH, J. D., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. I, Chap. 8. Academic Press, New York, 1955.
BIOCHEMISTRY
Thin-Layer
28, 313-317
Electrophoresis JOAO
Division
of
(1969)
of
Ribonucleotides
P. MONJARDINO
G%emistry and Bioehemistr~, Imperial Cancer Reseamh Lincoln’s Inn Fields, London WC??, England Received August
Fund,
21, 1968
Mild alkaline hydrolysis of RNA and subsequent electrophoresis of the resulting mixture of 2’,3’-nucleotides is now a standard technique in nucleic acid base composition analysis. The extension of its use to characterize RNA labeled with P32 under various conditions has made it an invaluable tool for the study of RNA metabolism of the cell. The method reported here makes possible the completion of a satisfactory electrophoretic separation of ribonucleotides (20-40 pg) on thin layers of cellulose in 90 min at 4OC using only conventional equipment. Base ratio determinations were simple and reproducible. MATERIALS
AND
METHODS
The samples of RNA used included yeast RNA (Sigma type xl), pig pituitary total RNA extracted by a modification of the hot phenol method described by Cline (1)) and nuclear and cytoplasmic pig pituitary RNA’s prepared using the same extraction procedure after isolation of the two fractions according to Hiatt (2) or Roberts et al. (3). Hydrolysis was carried out in 0.3 N KOH (1 mg/ml) at 37OC for 18 hr. The pH of the hydrolyzate was then adjusted to 7.0 by addition of perchloric acid and the precipitate of potassium perchlorate was spun down. The clear supernatant was then transferred into another tube and concentrated in a rotary evaporator before being applied to the plates. The glass plates used (5 X 20 cm) were washed as described previously (4) before being coated with the absorbent. The cellulose (MN 300, Macherey, Nagel & Co.) was washed according to De Fillipes (5) and the plates were coated 500 mp thick using a Dessaga spreader, dried in an over at 100°C, and stored in a 313
314
J. P. MONJARDINO
desiccator. The buffer used was 0.02 M citrate buffer (pH.3.5) freshly prepared prior to each run by diluting 50 times a stock solution made up by the addition of 3 vol 1 M citric acid and 1 vol 1 M trisodium citrate. The sample was applied to the plate 6 cm from one end as a band 1.0-1.5 cm wide. The plate was then immersed in a jar containing the buffer up to 2 or 3 cm below the spotted band and quickly placed in a Shandon electrophoresis tank in such a way that the origin was on the side of the negative electrode; 3MM Whatman wicks which had been dipped in buffer and folded in half were then applied to both ends and the current switched on as soon as the sample zone was wetted by convergence of both buffer fronts. A voltage of 650 V was usually applied (giving a voltage gradient of approximately 50 v/cm) and the current was about 3-5 mA. The separation was carried out at 4OC and was completed in 90 min. The plates were then dried under an infrared lamp and the bands located and penciled round under ultraviolet light (254 rnp) . For determination of base composition the bands and a blank (an area of cellulose from either side of the sample “pathway” of roughly the same size as the bands) were scraped off with a razor blade, transferred to polystyrene tubes containing 0.5 ml of 0.1 N HCl, and eluted overnight. The absorbent was centrifuged and the supernatant transferred to microcuvets for measurement of optical density at the appropriate wavelengths (CMP 279 rnp; AMP 257 rnp; GMP 257 mp; UMP 260 mp; Uvispek spectrophotometer). Conversion to pg nucleotide used millimolar extinction coefficients determined from commercial preparations (Sigma) of the corresponding 5’-nucleotides dissolved in 0.1 N HCl (AMP 15.5; CMP 13.1; GMP 11.6; UMP 10.7). Base composition of the same RNA samples was also determined by the thin-layer chromatography (TLC) method previously reported (4). RESULTS
AND
DISCUSSION
A complete separation of the hydrolyzate mixture into four bands was achieved after each run (Fig. 1). The bands were clearly seen under UV light and were identified by their ultraviolet absorption spectra (Fig. 2) after overnight elution in 0.1 N HCl; they were seen to correspond (starting from the origin) to 2’,3’-CMP, 2’,3’AMP, 2’,3’-GMP, and 2’,3’-UMP, respectively (Fig. 1). The GMP band was sometimes split in two, an observation also reported by Davidson and Smellie (6) and probably due to separation of the 2’ and 3’ isomers.
TLE
OF
Q
(+) 1 () 0 (a)
315
RIBONUCLEOTIDES
(b)
0
(cl
(-)
Cd)
FIG. 1. Thin-layer electrophoresis of 0.3 N KOH hydrolyzate of pig pituitary cytoplasmic ribonucleic acid. The bands starting from the origin are: (a) CMP, (b) AMP, (c) GMP, (d) UMP.
Recoveries of accurately measured amounts of commercial standards (5’-nucleotides from Sigma) were found to be approximately 90%. Base ratio determinations performed on cytoplasmic and nuclear pituitary pig RNA as well as on commercial yeast RNA samples were easy to carry out and reproducible (Table 1). There is also satisfactory agreement with the base compositions obtained by means of the TLC method previously reported (4) (Table 1). The differences found for nuclear RNA are probably explained by the different preparative methods used. The present system shows several advantages when compared with the established electrophoretic separation on paper. It allows for much smaller amounts of RNA to be analyzed-20-40 pg as opposed to 200-800 pg (7) -and the
0.7.
:
0.6 _ 05s
r:.a 0
0.3. \, .._.*
02.
.d
‘.
0 I-
*. ... ... *\ ‘.._ *\ ‘.__‘. -..:.
\
\.
.>.. .._ e:?:.;:.JL.., \
0
220
230
240
260
270
Wavelength,
ny
250
280
290
300
FIG. 2. UV spectra of nucleic acid components separated by TLE from 0.3 N KOH hydrolyzate of pig pituitary RNA: -.-. band (a), CMP (as in Fig. 1);. . . . band (b),AMP; -band (c), GMP; ---band (d), UMP.
316
J. P. MONJARDINO
Base Compositions
TABLE 1 of Pig Pituitary and Yeast RNA’s by TLCd and TLE* Methods (molar percentages)
source
f&Z!
Pig pituitary cytoplasm
Pig pituitary nuclei
G
A
C
U
Method
4
34.1 zt.41
17.2 zk.60
27.8 b.64
20.9 zk.75
TLCd
5
34.1 ~1~1.26
17.6 f.49
26.4 1.71
21.9 f1.43
TLEo
5
34.4 *1.03
18.8 zt1.23
26.0 dz.8
20.9 f.53
TLC’
17.5 f1.21
24.0 1.59
21.6 zk.77
TLE-
30.1 fl.1
25.3 fl.O
20.5 zk1.2
24.1 Al.2
TLCd
31.9 1.82
23.8 Al.1
19.5 fl.33
24.8 fl.1
TLEa
4 Yeast
14
36.9 ht.61
’
0 Present method. b Nuclear fraction prepared according to (2). = Nuclear fraction prepared according to (3). a Previously published (4).
separation is completed in 90 mm instead of the 18 hr for the paper system. Using the latter, the length of the run can be shortened by increasing the voltage gradient but this adds to the technical difficulty of counteracting the excess of heat being generated and calls for special cooling devices which make the whole apparatus cumbersome and expensive. Cur system, on the contrary, allows for a very rapid separation without any special cooling devices being required. SUMMARY
Electrophoresis on thin layers of cellulose of an alkaline hydrolyzate of RNA provides an easy and reproducible method for base ratio determinations. Separation of a nucleotide mixture (20-40 pg) is completed in 90 min at 4OC and requires only conventional equipment. ACKNOWLEDGMENT I wish to thank
Mrs. J. Harman
for her valuable
technical
assistance.
TLE OF RIBONUCLEOTIDES
317
REFERENCES 1. CLINE, M. J., J. Lab. Clin. Med. 68, 33 (1966). 2. HIATT, H. H., J. Mol. Biol. 5, 217 (1962). 3. ROBERTS, W. K., NEWMAN, J. F. E., AND RUECKERT, R. R., J. Mol. Biol. 15, 92 (1966). 4. MONJARDINO, J. P., Anal. B&hem. 21, 308 (1967). 5. DE FILLIPES, F. M., Science 144, 1350 (1964). 6. DAVIDSON, J. N., AND SMELLIE, R. M. S., Biochem. J. 52, 699 (1952). ‘7. SMITH, J. D., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. I, Chap. 8. Academic Press, New York, 1955.