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Fractionation with ethanol of nucleic acids from viroid-infected plants
ANALYTICAL
BIOCHEMISTRY
Fractionation ANTONIO *Departamento
134,479-482
(1983)
with Ethanol of Nucleic Acids from Viroid-Infected GRANELL,*
RICARDD
FLORES,~’
AND
VICENTE
CONEJERO*
de Bioquimica, ETS Ingenieros Agrbnomos, Universidad Politknica tlnstituto de Agroquimica y Tecnologia de Alimentos (CSIC), Valencia Received
May
Plants
de Valencia. 10, Spain
Spain;
and
10, 1983
A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 55, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids. KEY WORDS: viroid; nucleic acids; DNA; RNA.
Viroids are small naked RNAs that cause disease in some plants (1). The standard methods used for viroid purification have been adapted from those generally applied to nucleic acids. Initial steps of most of these methods include extraction of nucleic acids from infected tissues with water-saturated phenol followed by lithium chloride fractionation (2,3). In this way, preparations of nucleic acids are obtained which have as main components DNA, 4S, 58, and 7s cellular RNAs and viroid RNA. Intermediate purification steps remove most of the contaminant nucleic acids, and final purification of viroid RNA is accomplished by preparative electrophoresis in polyacrylamide gels (3,4). Nucleic acid precipitations with ethanol are repeated several times during the purification procedure and, as they are primarily used for concentration purposes, an excess of ethanol is added in order to precipitate all the nucleic acids. In this report we describe conditions for a fractionation of nucleic acids with ethanol ’ To whom all correspondence should be addressed. Instituto de Agroquimica y Tecnologia de Alimentos, Calle Jaime Roig 11, Valencia IO, Spain.
which permits the separation of viroid RNA from most of the accompanying DNA and 4s RNA.
MATERIALS
METHODS
Chemicals. Acrylamide, N, N’-methylenebisacrylamide, and N,N,N’,N’-tetramethylethylenediamine were purchased from Sigma. DNase I was from Worthington and proteinase K from Merck. Phenol for nucleic acid extraction was freshly redistilled. All other chemicals were of analytical grade or the best commercially available. Preparation of samples. Nucleic acids were extracted from Gynura aurantiaca plants infected with a severe strain of CEV.2 Onehundred grams of leaves with symptoms were processed according to a method reported previously which includes extraction with water-saturated phenol and removal of large ribosomal RNAs by precipitation with 2 M LiCl (2). The solution ( 10 ml) of nucleic acids which did not precipitate in 2 M LiCl was divided ’ Abbreviations
479
AND
used: CEV.
citrus
0003-2697183
exocortis
viroid.
$3.00
Copyright (c’ 19x3 hy Acadcmlc Press. Inc. A,, rights of rcpmduc,wn ,n anv tbrm rcwncd
480
GRANELL,
FLORES, AND CONEJERO
into 0.1 -ml aliquots which were treated as follows. Fractionation of nucleic acids with ethanol. To different aliquots of the previous solution, either 0.2,0.3,0.4,0.5, 1, or 3 vol of absolute ethanol were added with constant mixing. After standing overnight on an ice bath, the precipitated nucleic acids were recovered by centrifugation at 10,OOOgfor 10 min and the dried pellets resuspended in 0.05 ml of 10 mM TrisHCl, pH 7.2, containing 10 mM KC1 and 0.1 mM MgC12 (buffer A). In some cases the ethanol precipitation was repeated several times after dissolving the nucleic acid pellets in buffer A and making this solution 2 M in LiCl. Electrophoresis of nucleic acids. After the ethanol fractionation the nucleic acids were analyzed in polyacrylamide slab gels (130 X 100 X 2 mm). The electrophoresis was carried out under partially denaturing conditions as described previously (5) with the exception that the ratio of bisacrylamide to acrylamide was 20. The gels were run at 4°C with a constant current of 15 mA until the tracking dye xylene cyanole FE reached a distance of 45 mm from the origin. After electrophoresis the
gels were stained overnight with 0.01% of toluidine blue in water and the background removed with several changes of water. Densitometry of the stained wet gels was carried out with a Beckman CDS-200 densitometer provided with a 520-nm interference filter. An external standard (655544 Beckman) for calibration of the densitometer was used. RESULTS AND DISCUSSION
The nucleic acid fraction soluble in 2 M LiCl is composed mainly of DNA, 4S, 5S, and 7s RNAs which accompany the viroid RNA. Addition of 3 vol of ethanol per 1 vol of 2 M LiCl solution, as indicated in the original method (2), led to a complete precipitation of all nucleic acids (Fig. 1A, lane I). We observed that even the addition of 1 vol of ethanol led to very similar results (Fig. 1A, lane e). However, addition of smaller amounts of ethanol precipitated preferentially some nucleic acid species (Fig. 1A, lanes a to d). For our purposes of CEV purification, an optimum was found with 0.4 to 0.5 vol of ethanol (Fig. lA, lanes c and d). Under these conditions, densitometric quantitation of the electrophoretic pat-
DNA 7s CEb
5s
45
FIG. 1. Analysis of nucleic acids insoluble at different ethanol concentrations. Electrophoretic patterns (A) and densitometric tracings (B) of 2 M LiCl-soluble nucleic acids from CEV-infected Gynuru aurantiacu precipitated with (a) 0.2, (b) 0.3, (c) 0.4, (d) 0.5, (e) 1, and (f) 3 vol of ethanol per volume of nucleic acid solution. Sample of lane (g) was processed like the one of lane (f) unless before electrophoresis it was incubated at 30°C for 1 h with 25 &ml of DNase pretreated with proteinax K.
ETHANOL
FRACTIONATION
terns (Fig. 1B) showed that viroid precipitation was essentially complete, whereas DNA and 4s RNA, which account for more than the 95% of the total nucleic acids in the starting fraction, were reduced to less than the 15 and 25%, respectively, of the amounts precipitable with 3 vol of ethanol. In the case of DNA, the fractionation with 0.4 to 0.5 vol of ethanol was equivalent to a digestion for I h at 30°C with 25 &ml of DNase I pretreated with proteinase K to remove RNase traces (6) (Fig. 1A, lanes c, d, and g). Addition of less than 0.4 vol of ethanol, led to partial viroid recovery (Fig. 1A, lanes a and b). For routine work we chose 0.5 vol of ethanol which allowed us to perform the precipitation either at 0°C or alternatively at -25°C whereas with 0.4 vol of ethanol the solution froze at -25°C. When several cycles of ethanol precipitation were applied to the same sample (each time dissolving the nucleic acid pellets in buffer A and making this solution 2 M in LiCl) a further removal of DNA and 4s RNA was achieved. Figure 2 reveals that after repeating three times the precipitation with 0.5 vol of ethanol, the
c
1
2
OF
NUCLEIC
481
ACIDS
DNA and 4s RNA contents were reduced to about a 5% of the initial ones, whereas CEVRNA was recovered almost completely (Fig. 2). Other RNAs such as 5s and 7s RNAs, representing less than the 3% of the nucleic acids in the starting fraction, showed a behavior similar to viroid RNA (Figs. 1 and 2). It is interesting to point out that in some experiments in which the salt concentration was 0.1 M NaCl instead of 2 M LiCI, the selective precipitation of viroid RNA with 0.5 vol of ethanol did not occur (data not shown). These observations could be explained by assuming that the insolubility of CEV-RNA at low ethanol concentrations is a consequence of its high degree of secondary structure (4) which is stabilized by a high salt concentration. If this is the case, the method here presented could also be useful in the purification of double-stranded RNAs from complex nucleic acid mixtures. We think therefore, that the addition of 0.5 instead of 2-3 vol of ethanol per volume of solution of nucleic acids in 2 M LiCl, presents considerable advantages in viroid purification.
3
DNA
60
4s
DNA
IS
CEV
5s
4s
FIG. 2. Effect of repeated ethanol precipitations on the fractionation of nucleic acids. Electrophoretic patterns (A) and densitometric tracings (B) of 2 M LiCl-soluble nucleic acids from CEV-infected Gynuru aurnntiaca precipitated one (I), two (2), or three (3) times with 0.5 vol of ethanol per volume of nucleic acid solution. Sample of lane (c) was a control where all nucleic acids were precipitated by using 3 vol of ethanol.
482
GRANELL.
FLOW,
It permits the easy removal of most of the contaminant RNA and DNA (obviating the need for treatment with DNase which often has RNase traces). Consequently, preparative polyacrylamide gels can be loaded with samples enriched in viroid RNA. ACKNOWLEDGMENTS We are indebted to V. Moncholi, M. Climent, and R. Martinez for their technical assistanceand to Dr. J. Hansen for his critical reading of the manuscript. This work has been partially supported by the Comisi6n Asesora de Investigaci6n Cientica y Ttica and the Comite de Gestibn para la Exportacibn de Frutos Citricos of Spain.
AND CONEJERO
REFERENCES I. Diener, T. 0. ( 1979) Viroids and Viroid Diseases,pp. l-7, Wiley-Interscience, New York. 2. Semancik, J. S., and Weathers, L. G. (1972) Virology 47,456-460. 3. Diener, T. O., Hadidi, A., and Owens, R. A. (1977) in Methods in Virology (Maramorosch, K., and Koprowski, H., eds.), Vol. 6, pp. 185-217, Academic Press, New York. 4. Semancik, J. S., Morris, T. J., Weathers, L. G., Rodorf, B. F., and Keams, D. R. ( 1975) Virology 63, 160167. 5. Stinger, H. L., Ramm, K., Domdey, H., Gross, H. J., Hence, K., and Riesner, D. (1979) FEBS Lett. 99, 117-122. 6. Tullis, R. H., and Rubin, H. (1980) Anal. Biochem. 107,260-264.
BIOCHEMISTRY
Fractionation ANTONIO *Departamento
134,479-482
(1983)
with Ethanol of Nucleic Acids from Viroid-Infected GRANELL,*
RICARDD
FLORES,~’
AND
VICENTE
CONEJERO*
de Bioquimica, ETS Ingenieros Agrbnomos, Universidad Politknica tlnstituto de Agroquimica y Tecnologia de Alimentos (CSIC), Valencia Received
May
Plants
de Valencia. 10, Spain
Spain;
and
10, 1983
A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 55, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids. KEY WORDS: viroid; nucleic acids; DNA; RNA.
Viroids are small naked RNAs that cause disease in some plants (1). The standard methods used for viroid purification have been adapted from those generally applied to nucleic acids. Initial steps of most of these methods include extraction of nucleic acids from infected tissues with water-saturated phenol followed by lithium chloride fractionation (2,3). In this way, preparations of nucleic acids are obtained which have as main components DNA, 4S, 58, and 7s cellular RNAs and viroid RNA. Intermediate purification steps remove most of the contaminant nucleic acids, and final purification of viroid RNA is accomplished by preparative electrophoresis in polyacrylamide gels (3,4). Nucleic acid precipitations with ethanol are repeated several times during the purification procedure and, as they are primarily used for concentration purposes, an excess of ethanol is added in order to precipitate all the nucleic acids. In this report we describe conditions for a fractionation of nucleic acids with ethanol ’ To whom all correspondence should be addressed. Instituto de Agroquimica y Tecnologia de Alimentos, Calle Jaime Roig 11, Valencia IO, Spain.
which permits the separation of viroid RNA from most of the accompanying DNA and 4s RNA.
MATERIALS
METHODS
Chemicals. Acrylamide, N, N’-methylenebisacrylamide, and N,N,N’,N’-tetramethylethylenediamine were purchased from Sigma. DNase I was from Worthington and proteinase K from Merck. Phenol for nucleic acid extraction was freshly redistilled. All other chemicals were of analytical grade or the best commercially available. Preparation of samples. Nucleic acids were extracted from Gynura aurantiaca plants infected with a severe strain of CEV.2 Onehundred grams of leaves with symptoms were processed according to a method reported previously which includes extraction with water-saturated phenol and removal of large ribosomal RNAs by precipitation with 2 M LiCl (2). The solution ( 10 ml) of nucleic acids which did not precipitate in 2 M LiCl was divided ’ Abbreviations
479
AND
used: CEV.
citrus
0003-2697183
exocortis
viroid.
$3.00
Copyright (c’ 19x3 hy Acadcmlc Press. Inc. A,, rights of rcpmduc,wn ,n anv tbrm rcwncd
480
GRANELL,
FLORES, AND CONEJERO
into 0.1 -ml aliquots which were treated as follows. Fractionation of nucleic acids with ethanol. To different aliquots of the previous solution, either 0.2,0.3,0.4,0.5, 1, or 3 vol of absolute ethanol were added with constant mixing. After standing overnight on an ice bath, the precipitated nucleic acids were recovered by centrifugation at 10,OOOgfor 10 min and the dried pellets resuspended in 0.05 ml of 10 mM TrisHCl, pH 7.2, containing 10 mM KC1 and 0.1 mM MgC12 (buffer A). In some cases the ethanol precipitation was repeated several times after dissolving the nucleic acid pellets in buffer A and making this solution 2 M in LiCl. Electrophoresis of nucleic acids. After the ethanol fractionation the nucleic acids were analyzed in polyacrylamide slab gels (130 X 100 X 2 mm). The electrophoresis was carried out under partially denaturing conditions as described previously (5) with the exception that the ratio of bisacrylamide to acrylamide was 20. The gels were run at 4°C with a constant current of 15 mA until the tracking dye xylene cyanole FE reached a distance of 45 mm from the origin. After electrophoresis the
gels were stained overnight with 0.01% of toluidine blue in water and the background removed with several changes of water. Densitometry of the stained wet gels was carried out with a Beckman CDS-200 densitometer provided with a 520-nm interference filter. An external standard (655544 Beckman) for calibration of the densitometer was used. RESULTS AND DISCUSSION
The nucleic acid fraction soluble in 2 M LiCl is composed mainly of DNA, 4S, 5S, and 7s RNAs which accompany the viroid RNA. Addition of 3 vol of ethanol per 1 vol of 2 M LiCl solution, as indicated in the original method (2), led to a complete precipitation of all nucleic acids (Fig. 1A, lane I). We observed that even the addition of 1 vol of ethanol led to very similar results (Fig. 1A, lane e). However, addition of smaller amounts of ethanol precipitated preferentially some nucleic acid species (Fig. 1A, lanes a to d). For our purposes of CEV purification, an optimum was found with 0.4 to 0.5 vol of ethanol (Fig. lA, lanes c and d). Under these conditions, densitometric quantitation of the electrophoretic pat-
DNA 7s CEb
5s
45
FIG. 1. Analysis of nucleic acids insoluble at different ethanol concentrations. Electrophoretic patterns (A) and densitometric tracings (B) of 2 M LiCl-soluble nucleic acids from CEV-infected Gynuru aurantiacu precipitated with (a) 0.2, (b) 0.3, (c) 0.4, (d) 0.5, (e) 1, and (f) 3 vol of ethanol per volume of nucleic acid solution. Sample of lane (g) was processed like the one of lane (f) unless before electrophoresis it was incubated at 30°C for 1 h with 25 &ml of DNase pretreated with proteinax K.
ETHANOL
FRACTIONATION
terns (Fig. 1B) showed that viroid precipitation was essentially complete, whereas DNA and 4s RNA, which account for more than the 95% of the total nucleic acids in the starting fraction, were reduced to less than the 15 and 25%, respectively, of the amounts precipitable with 3 vol of ethanol. In the case of DNA, the fractionation with 0.4 to 0.5 vol of ethanol was equivalent to a digestion for I h at 30°C with 25 &ml of DNase I pretreated with proteinase K to remove RNase traces (6) (Fig. 1A, lanes c, d, and g). Addition of less than 0.4 vol of ethanol, led to partial viroid recovery (Fig. 1A, lanes a and b). For routine work we chose 0.5 vol of ethanol which allowed us to perform the precipitation either at 0°C or alternatively at -25°C whereas with 0.4 vol of ethanol the solution froze at -25°C. When several cycles of ethanol precipitation were applied to the same sample (each time dissolving the nucleic acid pellets in buffer A and making this solution 2 M in LiCl) a further removal of DNA and 4s RNA was achieved. Figure 2 reveals that after repeating three times the precipitation with 0.5 vol of ethanol, the
c
1
2
OF
NUCLEIC
481
ACIDS
DNA and 4s RNA contents were reduced to about a 5% of the initial ones, whereas CEVRNA was recovered almost completely (Fig. 2). Other RNAs such as 5s and 7s RNAs, representing less than the 3% of the nucleic acids in the starting fraction, showed a behavior similar to viroid RNA (Figs. 1 and 2). It is interesting to point out that in some experiments in which the salt concentration was 0.1 M NaCl instead of 2 M LiCI, the selective precipitation of viroid RNA with 0.5 vol of ethanol did not occur (data not shown). These observations could be explained by assuming that the insolubility of CEV-RNA at low ethanol concentrations is a consequence of its high degree of secondary structure (4) which is stabilized by a high salt concentration. If this is the case, the method here presented could also be useful in the purification of double-stranded RNAs from complex nucleic acid mixtures. We think therefore, that the addition of 0.5 instead of 2-3 vol of ethanol per volume of solution of nucleic acids in 2 M LiCl, presents considerable advantages in viroid purification.
3
DNA
60
4s
DNA
IS
CEV
5s
4s
FIG. 2. Effect of repeated ethanol precipitations on the fractionation of nucleic acids. Electrophoretic patterns (A) and densitometric tracings (B) of 2 M LiCl-soluble nucleic acids from CEV-infected Gynuru aurnntiaca precipitated one (I), two (2), or three (3) times with 0.5 vol of ethanol per volume of nucleic acid solution. Sample of lane (c) was a control where all nucleic acids were precipitated by using 3 vol of ethanol.
482
GRANELL.
FLOW,
It permits the easy removal of most of the contaminant RNA and DNA (obviating the need for treatment with DNase which often has RNase traces). Consequently, preparative polyacrylamide gels can be loaded with samples enriched in viroid RNA. ACKNOWLEDGMENTS We are indebted to V. Moncholi, M. Climent, and R. Martinez for their technical assistanceand to Dr. J. Hansen for his critical reading of the manuscript. This work has been partially supported by the Comisi6n Asesora de Investigaci6n Cientica y Ttica and the Comite de Gestibn para la Exportacibn de Frutos Citricos of Spain.
AND CONEJERO
REFERENCES I. Diener, T. 0. ( 1979) Viroids and Viroid Diseases,pp. l-7, Wiley-Interscience, New York. 2. Semancik, J. S., and Weathers, L. G. (1972) Virology 47,456-460. 3. Diener, T. O., Hadidi, A., and Owens, R. A. (1977) in Methods in Virology (Maramorosch, K., and Koprowski, H., eds.), Vol. 6, pp. 185-217, Academic Press, New York. 4. Semancik, J. S., Morris, T. J., Weathers, L. G., Rodorf, B. F., and Keams, D. R. ( 1975) Virology 63, 160167. 5. Stinger, H. L., Ramm, K., Domdey, H., Gross, H. J., Hence, K., and Riesner, D. (1979) FEBS Lett. 99, 117-122. 6. Tullis, R. H., and Rubin, H. (1980) Anal. Biochem. 107,260-264.