Journal of Biochemical and Biophysical Methods, 23 (1991) 189-192 © 1991 Elsevier Science Publishers B.V. 0165-022X/91/$03.50 ADONIS 0165022X9100092M
189
JBBM 00893
Short Note
A simple and rapid experimental protocol for studies of nucleic acids metabolism and their base composition A. Liras Facultad de Medicina, Universidad Aut6noma de Madrid, Spain (Received 19 February 1991) (Accepted 7 March 1991)
Summary A suitable, simple and rapid protocol for metabolic studies of nucleic acids and determining their base composition, using reversed-phase high-performance liquid chromatography is described. Modified classic methods of isolation of the nucleic acids fraction from a biological material, in our particular case Anemia sp., were used. Then analysis of their constituents and the incorporated radioactivity, after hydrolytic processes, was performed by high-performance liquid chromatography ~mder isocratic conditions, with 9 min total retention time. This method rhay be applied in several aspects of nucleic acids research, such as molecular cloning or metabolic and phylogenetic studies. Key words: HPLC; Nucleic acid metabolism; Base composition; Anemia
Introduction Nucleic acids metabolism is currently assessed in living cells by in vivo incorporation studies of exogenous labelled precursors and recovering the radioactivity incorporated into acid-soluble nucleotides or nucleic acids. On the other hand, nucleic acids base composition, which varies widely according to the biological material of origin, may be a crucial requisite in several studies of nucleic acids research, such as molecular cloning, metabolic aspects or phylogenetic studies (value of G + C content). A procedure for the study of nucleic acids metabolism and their base composition is described here, using reversed-phase high-performance liquid chromatograCorrespondence address: A. Liras, Ferraz 114, 4 ° D, 28008 Madrid, Spain.
190 phy, which has greatly facilitated the analysis of nucleotides, nucleosides and bases from biological materials, in our particular case under isocratic conditions. The protocol is based on a rapid and simple isolation of the nucleic acids fraction from Artemia sp. and then analysis of their free constituents released by a hydrolytic process and measuring of the incorporated radioactivity.
Material and Methods
Artemia cysts were obtained from San Francisco Bay Brand Co., Menlo Park, Ca, U.S.A. [U-14C]glycine was from The Radiochemical Centre (Amersham, U.K.). All nonradioactive standard purine bases and nucleotides were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Eagle's Minimal Essential Medium containing balanced salt solution was from Grand Island Biological Co. (Gibco). All other products were of analytical grade. 40-hour-old Artemia nauplii were obtained from dried embryos treated as described [1], except for the salt concentration of the growth medium which was diluted 6-fold, and incubated for in vivo incorporation studies under the same conditions as described in Ref. 2 in the presence of [U-~4C]glycine (specific activity approx. 56 Ci/mol), at a final concentration of 0.15 mM. After 3 h of incubation at 30 ° C under sterile conditions, nauplii were collected by filtration and washed with 10 mM NaHCO 3. Acid-soluble extracts were obtained by standard methods involving homogenization in 0.5 N perchloric acid at 4 o C. The acid-insoluble material was washed with cold ethanol, delipidated by extraction with ethanol/ether and extracted with hot 10% NaCI to obtain the nucleic acids fraction as previously described [3]. RNA and DNA were precipitated by cold ethanol at - 80 ° C for 60 min and redissolved in water. RNA was selectively hydrolized in 0.3 M KOH at 37 °C overnight and undigested DNA separated by acid precipitation with HCI to pH 2.0 and centrifugation. The neutralized supernatant containing 2'- and 3'-RNA nucleotides was analyzed by reversed-phase HPLC, using a Waters liquid chromatograph (Waters Assoc., Milford, MA, U.S.A.) equipped with a 6000-A pump, a U6K injector and a 440 UV detector at 254 nm, on a Nova-Pak C~8 (150 x 4 mm, 5 /zm particle) column from Waters Assoc. The precipitated DNA was washed with cold ethanol, redissolved in water and hydrolized in 98% formic acid at 180 ° C for 1 h [4]. After hydrolysis the acid was eliminated under vacuum at 50°C and the residue redissolved in the buffer used to elute the HPLC column Polygosil C~8 (200 X 4 mm, 5 ~m particle) from Macherey Nagel for the analysis of free bases released.
Results and Discussion
A simple and rapid protocol for metabolic studies of nucleic acids and for determining their base composition is described. Modified classic methods of isolation of the nucleic acids fraction from the biological material, in this case from
191
3'UMP
A)
I 2'UMP I / n 3'GMP
il
Kli
CMP
\
BI
!I/ 'I
j
I I
I I
I~
i
it'
III
II
I I
CYTOSINE
t[
LI~
LtlllUl li', 1 -
- -
[
, 1 ~_j
_
I0
RETENTION
-
0
TIME (rain)
Fig. 1. Chromatographic ( ) and radioactivity ( - - - - - - ) profiles of isocratic reverse-phase H P L C analysis of metabolically labelled R N A nucleotides and D N A bases after incubation of 40-hour-old
Artemia nauplii with [UJ4C]glycine.A: chromatographicprofile of alkali-derived RNA nucleotides on
Nova-Pak C18 column, KH2PO4 0.1 M, pH 4.0, 2.5% methanol buffer and flow rate 1.5 ml/min; B: chromatographic profile of formic-acid-derivedDNA bases on PolygosilC18 column, KH2PO 4 10 mM, pH 3.7, 5% methanol buffer and flow rate 1.0 ml/min.
crustacean Artemia sp., were used and their constituents analyzed using reversedphase high-performance liquid chromatography under isocratic conditions. The incorporation of [u-lac]glycine as purine-labelled precursor, into alkali-derived R N A nucleotides and formic hydrolysis-derived D N A bases, is shown in Fig. 1, which represents the chromatographic profile of 2'- and 3'-RNA nucleotides (A) and free bases from D N A (B), and the radioactivity incorporated by Artemia sp. nauplii. Peaks were identified by their retention times and coelution with authentic nucleotide or base standards, and the molar concentration of each nucleic acids constituent, which is necessary for calculating the percentage of base composition, was determined by comparison with the standards in which individual concentrations were fixed by spectrophotometric titration. Chromatographic fractions were collected every 20 s and analyzed for radioactivity incorporated into RNA nucleotides and D N A bases by liquid scintillation counting. The base composition of R N A of Artemia nauplii, using this experimental protocol, represents 21% for cytosine, 17% for uracil, 32% for guanine and 30% for adenine, and the G + C content was 53%. With respect to this value of G + C content obtained, we may consider the crustacean Artemia to be at a low level in the phylogenetic scale [5].
192
Because the composition of nucleic acids hydrolysates is relatively simple compared with the composition of acid-soluble extracts which contain a great diversity of interfering low-molecular-weight substances, this experimental protocol, with a rapid and simple isolation of nucleic acids components and a short analysis time using HPLC, 9 min total retention time, and under isocratic conditions without using gradient elution, is very appropriate for these studies. The rapidity and the excellent resolution allow the detection of labelled components of nucleic acids, and an easy determination of the percentage distribution of radioactivity in nucleotide metabolism studies by in vivo incorporation of glycine as purine precursor and/or bicarbonate as purine and pyrimidine precursor, the molar concentration, base composition and the specific activity. In conclusion, this method may be applied to related areas of nucleic acids research, such as molecular cloning or metabolic and phylogenetic studies.
References 1 Llorente, P. and Ruiz-Cfirdaba, A. (1987) Carbamylphosphate synthetase activities during Artemia development. In: Decleir, W., Moens, L., Slegers, H., Jaspers, E. and Sorgeloos, P. (Eds.) Artemia Research and its applications, Universa Press, Belgium, pp. 79-92. 2 Llorente, P., Carratalfi, M., Liras, A. and Rotll~in, P. (1987) De novo purine biosynthesis in developing Artemia nauplii. In: Decleir, W., Moens, L., Slegers, H., Jaspers, E. and Sorgeloos, P. (Eds.) Artemia Research and its applications, Universa Press, Belgium, pp. 243-252. 3 Tyner, E.P., Heidelberger, C. and Le Page, G.A. (1953) Intracellular distribution of radioactivity in nucleic acid, nucleotides and proteins following simultaneous administration of p32 and glycine-2-C 14. Cancer Res. 13, 186-203. 4 Wyatt, G.R. and Cohen, S.S. (1953) The bases of the nucleic acids of some bacterial and animal viruses: the occurrence of 5-hydroxymethylcytosine. Biochem, J. 55, 774-782. 5 Davidson, J.N. (1972) The structure of RNA. In: The biochemistry of the nucleic acids, Chapman and Hall, Norfolk, pp. 106-107.
189
JBBM 00893
Short Note
A simple and rapid experimental protocol for studies of nucleic acids metabolism and their base composition A. Liras Facultad de Medicina, Universidad Aut6noma de Madrid, Spain (Received 19 February 1991) (Accepted 7 March 1991)
Summary A suitable, simple and rapid protocol for metabolic studies of nucleic acids and determining their base composition, using reversed-phase high-performance liquid chromatography is described. Modified classic methods of isolation of the nucleic acids fraction from a biological material, in our particular case Anemia sp., were used. Then analysis of their constituents and the incorporated radioactivity, after hydrolytic processes, was performed by high-performance liquid chromatography ~mder isocratic conditions, with 9 min total retention time. This method rhay be applied in several aspects of nucleic acids research, such as molecular cloning or metabolic and phylogenetic studies. Key words: HPLC; Nucleic acid metabolism; Base composition; Anemia
Introduction Nucleic acids metabolism is currently assessed in living cells by in vivo incorporation studies of exogenous labelled precursors and recovering the radioactivity incorporated into acid-soluble nucleotides or nucleic acids. On the other hand, nucleic acids base composition, which varies widely according to the biological material of origin, may be a crucial requisite in several studies of nucleic acids research, such as molecular cloning, metabolic aspects or phylogenetic studies (value of G + C content). A procedure for the study of nucleic acids metabolism and their base composition is described here, using reversed-phase high-performance liquid chromatograCorrespondence address: A. Liras, Ferraz 114, 4 ° D, 28008 Madrid, Spain.
190 phy, which has greatly facilitated the analysis of nucleotides, nucleosides and bases from biological materials, in our particular case under isocratic conditions. The protocol is based on a rapid and simple isolation of the nucleic acids fraction from Artemia sp. and then analysis of their free constituents released by a hydrolytic process and measuring of the incorporated radioactivity.
Material and Methods
Artemia cysts were obtained from San Francisco Bay Brand Co., Menlo Park, Ca, U.S.A. [U-14C]glycine was from The Radiochemical Centre (Amersham, U.K.). All nonradioactive standard purine bases and nucleotides were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Eagle's Minimal Essential Medium containing balanced salt solution was from Grand Island Biological Co. (Gibco). All other products were of analytical grade. 40-hour-old Artemia nauplii were obtained from dried embryos treated as described [1], except for the salt concentration of the growth medium which was diluted 6-fold, and incubated for in vivo incorporation studies under the same conditions as described in Ref. 2 in the presence of [U-~4C]glycine (specific activity approx. 56 Ci/mol), at a final concentration of 0.15 mM. After 3 h of incubation at 30 ° C under sterile conditions, nauplii were collected by filtration and washed with 10 mM NaHCO 3. Acid-soluble extracts were obtained by standard methods involving homogenization in 0.5 N perchloric acid at 4 o C. The acid-insoluble material was washed with cold ethanol, delipidated by extraction with ethanol/ether and extracted with hot 10% NaCI to obtain the nucleic acids fraction as previously described [3]. RNA and DNA were precipitated by cold ethanol at - 80 ° C for 60 min and redissolved in water. RNA was selectively hydrolized in 0.3 M KOH at 37 °C overnight and undigested DNA separated by acid precipitation with HCI to pH 2.0 and centrifugation. The neutralized supernatant containing 2'- and 3'-RNA nucleotides was analyzed by reversed-phase HPLC, using a Waters liquid chromatograph (Waters Assoc., Milford, MA, U.S.A.) equipped with a 6000-A pump, a U6K injector and a 440 UV detector at 254 nm, on a Nova-Pak C~8 (150 x 4 mm, 5 /zm particle) column from Waters Assoc. The precipitated DNA was washed with cold ethanol, redissolved in water and hydrolized in 98% formic acid at 180 ° C for 1 h [4]. After hydrolysis the acid was eliminated under vacuum at 50°C and the residue redissolved in the buffer used to elute the HPLC column Polygosil C~8 (200 X 4 mm, 5 ~m particle) from Macherey Nagel for the analysis of free bases released.
Results and Discussion
A simple and rapid protocol for metabolic studies of nucleic acids and for determining their base composition is described. Modified classic methods of isolation of the nucleic acids fraction from the biological material, in this case from
191
3'UMP
A)
I 2'UMP I / n 3'GMP
il
Kli
CMP
\
BI
!I/ 'I
j
I I
I I
I~
i
it'
III
II
I I
CYTOSINE
t[
LI~
LtlllUl li', 1 -
- -
[
, 1 ~_j
_
I0
RETENTION
-
0
TIME (rain)
Fig. 1. Chromatographic ( ) and radioactivity ( - - - - - - ) profiles of isocratic reverse-phase H P L C analysis of metabolically labelled R N A nucleotides and D N A bases after incubation of 40-hour-old
Artemia nauplii with [UJ4C]glycine.A: chromatographicprofile of alkali-derived RNA nucleotides on
Nova-Pak C18 column, KH2PO4 0.1 M, pH 4.0, 2.5% methanol buffer and flow rate 1.5 ml/min; B: chromatographic profile of formic-acid-derivedDNA bases on PolygosilC18 column, KH2PO 4 10 mM, pH 3.7, 5% methanol buffer and flow rate 1.0 ml/min.
crustacean Artemia sp., were used and their constituents analyzed using reversedphase high-performance liquid chromatography under isocratic conditions. The incorporation of [u-lac]glycine as purine-labelled precursor, into alkali-derived R N A nucleotides and formic hydrolysis-derived D N A bases, is shown in Fig. 1, which represents the chromatographic profile of 2'- and 3'-RNA nucleotides (A) and free bases from D N A (B), and the radioactivity incorporated by Artemia sp. nauplii. Peaks were identified by their retention times and coelution with authentic nucleotide or base standards, and the molar concentration of each nucleic acids constituent, which is necessary for calculating the percentage of base composition, was determined by comparison with the standards in which individual concentrations were fixed by spectrophotometric titration. Chromatographic fractions were collected every 20 s and analyzed for radioactivity incorporated into RNA nucleotides and D N A bases by liquid scintillation counting. The base composition of R N A of Artemia nauplii, using this experimental protocol, represents 21% for cytosine, 17% for uracil, 32% for guanine and 30% for adenine, and the G + C content was 53%. With respect to this value of G + C content obtained, we may consider the crustacean Artemia to be at a low level in the phylogenetic scale [5].
192
Because the composition of nucleic acids hydrolysates is relatively simple compared with the composition of acid-soluble extracts which contain a great diversity of interfering low-molecular-weight substances, this experimental protocol, with a rapid and simple isolation of nucleic acids components and a short analysis time using HPLC, 9 min total retention time, and under isocratic conditions without using gradient elution, is very appropriate for these studies. The rapidity and the excellent resolution allow the detection of labelled components of nucleic acids, and an easy determination of the percentage distribution of radioactivity in nucleotide metabolism studies by in vivo incorporation of glycine as purine precursor and/or bicarbonate as purine and pyrimidine precursor, the molar concentration, base composition and the specific activity. In conclusion, this method may be applied to related areas of nucleic acids research, such as molecular cloning or metabolic and phylogenetic studies.
References 1 Llorente, P. and Ruiz-Cfirdaba, A. (1987) Carbamylphosphate synthetase activities during Artemia development. In: Decleir, W., Moens, L., Slegers, H., Jaspers, E. and Sorgeloos, P. (Eds.) Artemia Research and its applications, Universa Press, Belgium, pp. 79-92. 2 Llorente, P., Carratalfi, M., Liras, A. and Rotll~in, P. (1987) De novo purine biosynthesis in developing Artemia nauplii. In: Decleir, W., Moens, L., Slegers, H., Jaspers, E. and Sorgeloos, P. (Eds.) Artemia Research and its applications, Universa Press, Belgium, pp. 243-252. 3 Tyner, E.P., Heidelberger, C. and Le Page, G.A. (1953) Intracellular distribution of radioactivity in nucleic acid, nucleotides and proteins following simultaneous administration of p32 and glycine-2-C 14. Cancer Res. 13, 186-203. 4 Wyatt, G.R. and Cohen, S.S. (1953) The bases of the nucleic acids of some bacterial and animal viruses: the occurrence of 5-hydroxymethylcytosine. Biochem, J. 55, 774-782. 5 Davidson, J.N. (1972) The structure of RNA. In: The biochemistry of the nucleic acids, Chapman and Hall, Norfolk, pp. 106-107.
