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In vitro labeling of polynucleotides by photoreduction
174
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96836
I N V I T R O L A B E L I N G OF P O L Y N U C L E O T I D E S BY P H O T O R E D U C T I O N V I V I E N F. L E E * AND M I L T O N P. G O R D O N
Department o/Biochemistry, University of Washington, Seattle, Wash. 98105 (U.S.A.) (Received D e c e m b e r I4th, I97 o)
SUMMARY
I. Treatment with NaB3H, in the presence of ultraviolet irradiation is a rapid and economical method of introducing 8H into DNA and RNA. The reaction was modified into a usable laboratory procedure. Specific activities of up to 8700 counts/rain per #g DNA were easily attained. 2. The reaction products were subjected to physicochemical analysis. The results of the present study suggest that the labeled DNA shows some very minor degradation and lowered thermal stability particularly at high temperatures; otherwise its general physical properties are not detectably altered. Therefore, DNA so labeled can be used for physical and hybridization studies.
INTRODUCTION
Many studies of polynucleotides require that these polymers be radioactive. At present, the most widely used means of obtaining radioactive polynucleotides is the in vivo method of labeling. However, where the organisms are large or rare, or where rates of nucleic acid biosynthesis preclude efficient labeling in an entire organism, one of two alternatives must be used.The alternate most often employed, tissue culture, retains m a n y of the disadvantages of other in vivo procedures. The second alternate is the in vitro labeling of isolated polynucleotides. Most of the documented procedures of in vitro labeling either use excessive amounts of radioactive isotopes 1 or leave the polynucleotides degraded z,3. A methylation procedure has been used for RNA with some degree of success 4 although this method does not seem to be useful for DNA z. Hence, a versatile and economical method that can introduce radioisotopes with minimal alterations of the isolated polymers would be of considerable value. When polynucleotides are treated with sodium borohydride in the presence o~ ultraviolet irradiation, a selective reduction of the pyrimidines occurs 5. The reactivity of the bases, specifically thymine in DNA 6 and uracil in RNA s, suggests a useful method for introducing radioactive isotopes into polynucleotides. This paper discusses the application of the reaction in labeling DNA and RNA and examines the physicochemical properties of the modified DNA product. * S u b m i t t e d in p a r t i a l fulfillment of t h e r e q u i r e m e n t s for a M a s t e r of Science Degree from t h e D e p a r t m e n t of B i o c h e m i s t r y U n i v e r s i t y of W a s h i n g t o n , Seattle, W a s h . 98105, U.S.A.
Bioehim. Biophys. Acta, 238 ~t97 I) 174-.179
LABELING OF POLYNUCLEOTIDESBY PHOTOREDUCTION
175
MATERIALS DNA's from sea urchin and Bacillus subtilis were the gift of Mrs. Margaret Farquhar of this department. Calf thymus DNA was obtained from Miles Chemical Co. B. subtilis E14ClDNA was the gift of Dr. Mary-Dell Chilton of this department. NaB3H4 was obtained from Amersham-Searle Corp. Hydroxylapatite was obtained from Clarkson Chemical Co. Bovine serum albumin was obtained from Armour Pharmaceutical Co. All other chemical compounds were standard ACS reagent grade. Buffer was o.15 M NaCl-o.oI5 M sodium citrate, pH7.2. An ultraviolet lamp, UVS-I2, was obtained from Ultraviolet Products, Inc., San Gabriel, Calif., U.S.A.
METHODS AND RESULTS
Photoreduction o/ polynucleotides A typical in vitro labeling experiment is the following: A sample of native DNA ( o . I - 0 . 5 m g / m l ) o r RNA (0.5 mg/ml) was dissolved in o.o165 M sodium borate buffer, p H 9.0. Nitrogen gas was bubbled through the solution to displace oxygen which might interfere with the reaction. An aliquot was then transferred to a quartz cuvette and gently stirred throughout the reaction. A 5- to Io-fold molar excess of NaB3HI (relative to DNA thymine) freshly dissolved in the borate buffer was added to the solution of polynucleotides. This solution was immediately placed behind a i - c m quartz cuvette containing 20 % acetic acid solution which served to filter ultraviolet light below 2400 A. The lamp charactelistics were such that a distance of 17.8 cm from the reaction cuvette gave a dosage of 347 ° ergs/mm 2 per rain at 253.7 nm. After 15 min of irradiation the reaction was stopped by the addition of o.I ml of o.I M sodium citrate, p H 5.3, for every I.O ml of nucleic acid. The excess NaBSHI decomposed with vigorous bubbling. The nucleic acids were then either directly dialyzed versus 0.05 M sodium citrate, p H 5.3, or alcohol precipitated prior to dialysis. After 4 or 5 changes of dialysis buffer when the radioactivity of the dialysate was reduced to background level, the nucleic acids were transferred to o.15 M NaC1O.Ol5 M sodium citrate buffer and subsequently stored at --20 ° in the same buffer. All photoreductions were run in the fume hood. The concentrations of the labeled nucleic acids were measured on a Cary 15 spectrophotometer, and their radioactivities measured in the Beckman CPM-Ioo liquid scintillation system.
Labeling o/ di//erent polynucleotide preparations under various conditions Experiments were performed to test the effects of certain reaction parameters on the extent of labeling. The concentrations of all DNA solutions were 0.595 mg/ml in o.o165 M borate buffer, p H 9.0. For denaturation, the samples were placed in boiling water for IO min. The NaBSH4 stock solution was 0.276 M in the alkaline borate buffer, and this solution was used within I rain after it was made. Identical aliquots containing 2.76/zmoles wele added to all the nucleic acid solutions for photoleduction. All samples were irradiated simultaneously. Irradiation periods were o, 2, 5, IO, 15, 30 and 60 rain for native DNA, and IO and 30 min for denatured DNA. For one sample of DNA that received no irradiation, reduction was allowed to pro-
Biochim. Biophys. Acta, 238 (i97 r) 174-179
176
v.F.
LEE, M. P. GORDON
ceed for 3o min. The RNA solution was I mg/ml and the irradiation time was 15 rain. The results of these experiments are shown in Fig. I.
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Fig. I. 8H i n c o r p o r a t i o n as a f u n c t i o n of irradiation t i m e . All D N A r e d u c t i o n s were r u n a t a ratio of i m M t h y m i n e to 5.3 m M NaBSH4. T h e R N A r e d u c t i o n w a s r u n at a ratio of 0.89 m M uracil to 5.3 mY[ NaBSH4. (~)-O, n a t i v e D N A , p h o t o r e d u c e d ; /x, n a t i v e D N A , r e d u c e d w i t h o u t i r r a d i a t i o n ; IN, d e n a t u r e d D N A , p h o t o r e d u c e d ; × , R N A , p h o t o r e d u c e d . Fig. 2. Sucrose g r a d i e n t s e d i m e n t a t i o n of calf t h y m u s D N A . iN- - -El, unlabeled, u n t r e a t e d D N A , a b s o r b a n c e p a t t e r n ; O - O , i n vitro labeled D N A , a b s o r b a n c e p a t t e r n ; A - & , in vitro labeled D N A , radioactivity pattern.
Band sedimentation o/the labeled DNA Neutral sucrose gradient sedimentation. Photoreduced calf thymus DNA (irradiated 15 rain), o.500 mg per 0. 4 ml, was layered on a 4.6 ml linear sucrose gradient, 6-3 ° %. The gradient was centrifuged at 48 ooo rev./min (approx. 2 o o o o o × g ) for 75 min at 15 ° in the SW 5o.1 rotor in the Beckman L2-65 B centrifuge. Io-drop fractions were then collected and their absorbances measured at 260 nm; 0.8 ml of distilled water was added to each fraction. The fractions were then dialyzed against three changes of O.Ol5 M NaCl-o.ooI 5 M sodium citrate buffer. In order to determine the 3H content of each fraction, 0.2 ml bovine serum albumin (I mg/ml) was added followed b y 1.2 ml of a 50 % trichloroacetic acid solution. After IO min, the solutions were filtered through W h a t m a n GF/C filters which were then dried and counted in IO ml of toluene-liquiflor scintillation fluid. A sample of untreated calf thymus DN A was also subjected to band sedimentation under the same conditions. Fig. 2 shows the patterns obtained in this experiment. Alkaline sucrose gradient sedimentation. A mixture of equal amounts of unlabeled and labeled calf thymus DNA (irradiated 15 min), 12o/~g total in 0. 4 ml solution, was layered on a 4.6-ml linear alkaline sucrose gradient (6-30 % sucrose in 0. 7 M NaCl-o.3 M NaOH). The tubes were then centrifuged at 49 ooo rev./min (approx. 225 ooo ×g) fol 5 h at 25 ° in the SW 5o.1 rotor in the Beckman L2-65B centrifuge. At the end of the centrifugation, Io-drop fractions were again collected and treated as above. The data from this experiment are shown in Fig. 3. Heat stability o! the labeled DNA Capillaries containing 5 °/~1 aliquots of sheared labeled B. subtilis DNA (2oo Biochim. Biophys, Acta, 238 (1971) i 7 4 - I 7 9
ABELING OF POLYNUCLEOTIDES BY PHOTOREDUCTION
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Fig. 3. Alkaline sucrose gradient sedimentation of calf thymus DNA. The gradient contained cold and labeled material in the ratio of i : I. O - Q ) , absorbance pattern; &-&, radioactivity pattern. Fig. 4. Heat stabilities of labeled B. subtilis DNA. - - , specific activities of in vitro labeled DNA; - - -, specific activities of in vivo labeled [x4C]DNA. The horizontal lines indicate samples which had not been heated. Q), denatured DNA, incubated at 60°; A native DNA, incubated at 70°; El, native DNA, incubated at 80°; 0 , native DNA, incubated at 9o°.
/zg/ml, 13o c o u n t s / m i n per #g, in o.15 IV[NaCl-o.oI5 M sodium citrate buffer) were sealed a n d t r e a t e d in the following m a n n e r : For the 60 ° heat s t a b i l i t y study, the capillaries were kept at IOO° for IO min before being transferred to a 60 ° b a t h a n d i n c u b a t e d for as long as 23 h. I n the heat s t a b i l i t y studies carried out at 7 o°, 80 °, a n d 9 °0 , the samples were i n c u b a t e d for different lengths of time w i t h o u t prior denaturation. All samples were r u n in duplicate. A t the end of the i n c u b a t i o n periods, the capillaries were opened a n d their contents diluted i n 6 m l of 0.5 M phosphate buffer, p H 7.2. The r a d i o a c t i v i t y of the trichloroacetic acid-precipitable m a t e r i a l was t h e n determined. Samples of sheared 14C-labeled B. subtilis D N A were t r e a t e d identically. Fig. 4 shows the heat stabilities of the various samples. M e l t i n g curves o / t h e labeled D N A Melting curves of cold a n d labeled D N A were o b t a i n e d s i m u l t a n e o u s l y using the Gilford 20o0 spectrophotometer. All m e a s u r e m e n t s were made in o.15 M NAC1o.o15 M sodium citrate buffei. The Tm's a n d hyperchromicities at 260 n m are given in Table I.
TABLEI MELTING
C U R V E D A T A F R O M in
DNA
LABELED
NaBSH4 Irradiation specific activity time (min) (mC/mg)
Sea urchin
-
-
3.7 157.o Calf thymus
oitro
-
-
3.7 3.7
DNA DNA T,. specific activity (counts/rain per /~g)
Hyperchromicity at 260 nm
-15 io
o 260 87oo
83.8 83.3 83. 3
o.261 o.268 o.268
-IO 60
o 203 191
85.5 85.2 85. 3
0.345 0.350 0.363
Biochim. Biophys. Acta, 238 (i97 I) 174-179
178
V . F . LEE, M. P. GORDON
Renaturation kinetics o/ the labeled D N A as measured on hydroxylapatitd Photoreduced B. subtilis DNA, specific activity 13o counts/min per #g, was dissolved in o.15 M NaCl-o.oI 5 M sodium citrate buffer at a concentration of 200 #g/ ml or co = 0.606" lO -3 moles nucleotide per l. The solutions were then sheared in a French pressure cell at IO ooo-12 ooo lb/inch 2. Aliquots of 5oF1 were sealed in capillary micropipettes. The samples were denatured at ioo ° for IO min, then quickly transferred to a 60 ° bath. At intervals, samples were removed for measurement of renaturation by hydroxylapatite column chromatography. A reference sample of in vivo labeled B. subtilis DNA was treated identically. For this control sample, the initial co was 1.52- lO -3 moles nucleotide per 1. The results are shown in Fig. 5.
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DISCUSSION
The application of the photoreduction reaction to polynucleotides is promising in its convenience, its economy, and its relative safety. The alterations in the DNA produced by ultraviolet irradiation and incorporation of 3H produce minimal changes in the physical-chemical properties. It was found that the optimum concentration of native DNA was about 0. 5 mg/ml with a 5- to Io-fold molar excess of NaBSH4 relative to the thymine content. Higher concentrations of native DNA solution resulted in lower reaction rates probably due to the increased viscosity. The use of sheared or denatured DNA at higher concentrations would bypass the problem of viscosity. The results in Fig. i indicate that incorporation reaches a plateau after IO mill. Since photoreduction is known to proceed to ring cleavage, and in view of ultraviolet-induced lesions, most photoreactions were allowed to proceed only lO-15 min. It was estimated that one out of sixty thymine moieties had been altered, assuming one 3H substituent per base modified. Adjusting this value to the 58 % ( A + T ) content of calf thymus DNA s, the number of base pairs altered is o.97 °/o. This must be considered an upper limit since it is possible that there is more than one 3H per reduced base. Denatured DNA gave 25-35 % higher incorporation under the same conditions. RNA also showed comparable incorporation. Since some 3H was also incorporated without irradiation Biochim. Biophys. Acta, 238 (1971) 174-179
LABELING OF POLYNUCLEOTIDES BY PHOTOREDUCTION
179
(see Fig. I), a second reaction other than photoreduction could be occurring simultaneously. The sucrose gradient band sedimentation of the labeled DNA was a clear indication that the radioactivity was incorporated throughout the DNA (see Fig. 2). However, the alkaline sucrose gradient patterns (Fig. 3) of the absorbance and radioactivity suggest that minor degradation of the DNA has occurred. Compared to the in vivo labeled E14C]DNA, the photoreduced samples showed lowered thermal stabilities at high temperatures. However, the stability at 60 ° indicates that the photoreduced DNA is useful for a variety of hybridization studies. A 1. 5 % mismatching of bases results in a lowering of the T m by I ° (ref. 9)If the lowered T,,'s (Table I) are truly indicative of base mispairing and not due to technical limitations, an upper limit of 0.8 % of base mismatching is indicated. This number is in agreement with the calculations obtained for the number of base pairs altered, 0.97 %. The widths of the melting transitions were found to be essentially unchanged in the photoreduced samples. As measured by hydroxylapatite chromatography, the renaturation properties of the in vitro and in vivo labeled DNA's are identical (Fig. 6). A published value of cot½ = 5 for B. subtilis 1° was measured by absorption spectroscopy. Renaturation rates are usually 2-fold faster when measured by the hydroxylapatite method as compared to the optical method since the former measures the fraction of fragments reassociated, while the latter measures the total strand length reassociated 9. Allowing for this, our value of Cot~ = 3 compares favorably with the adjusted value of 2.5 for B. subtilis, measured under similar salt and temperature conditions. These results are in agreement with the stability of [3H]DNA at 60 °. It was recently brought to our attention that Dr. L. Kirkegaard and coworkers at the University of Wisconsin have prepared ZH-labeled tRNA, mRNA and rRNA b y procedures similar to those described here n.
ACKNOWLEDGMENTS
It is a pleasure to thank Dr. Brian J. MCCARTHY for his interest and helpfulness. This work was supported by funds from the U.S. Atomic Energy Commission, the National Science Foundation, and the Graduate School Research Fund, University of Washington. REFERENCES I 2 3 4 5 6 7 8 9 Io II
E. BORENFREUND, M. S. ROSENKRA~IZ AND A. BENDICH, J. Mol. Biol., i (1959) 195. F. ASCOLI AND F. IV[. KAHAN, J. Biol. Chem., 24I (1966) 428. D. G. SEARCH, Biochim. Biophys. Acta, 166 (1968) 36o. K. D. SMITH, J. L. ARMSTRONG AND B. J. McCARTHY, Biochim. Biophys. Acta, 142 (1967) 323. P- CERUTTI, K. IKEDA AND B. WITKOP, J. Am. Chem. Soc., 87 (1965) 2505. C-. BALLE, P. CERUTTI AND B. WITKOP, J. Am. Chem. Sot., 88 (1966) 3946. G. BERNARDI, Nature, 2o6 (1965) 779. C. LONG, Biochemists' Handbooh, D. V a n N o s t r a n d Co., P r i n c e t o n , N.J., 1961, p. 199. R. J. BRITTEN AND D. KOHNE, Science, 161 (1968) 529. B. L. McCoNAUGHY, C. D. LAIRD AND B. J. McCARTHY, Biochemistry, 8 (I969) 3289. L. KIRKEGAARD, P h . D. Thesis, U n i v e r s i t y of Wisconsin, 1969. C o m m u n i c a t e d b y Dr. R. M. BOCK, U n i v e r s i t y of W i s c o n s i n , Madison, ~risc.
Biochim. Biophys. Acta, 238 (1971) 174-179
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96836
I N V I T R O L A B E L I N G OF P O L Y N U C L E O T I D E S BY P H O T O R E D U C T I O N V I V I E N F. L E E * AND M I L T O N P. G O R D O N
Department o/Biochemistry, University of Washington, Seattle, Wash. 98105 (U.S.A.) (Received D e c e m b e r I4th, I97 o)
SUMMARY
I. Treatment with NaB3H, in the presence of ultraviolet irradiation is a rapid and economical method of introducing 8H into DNA and RNA. The reaction was modified into a usable laboratory procedure. Specific activities of up to 8700 counts/rain per #g DNA were easily attained. 2. The reaction products were subjected to physicochemical analysis. The results of the present study suggest that the labeled DNA shows some very minor degradation and lowered thermal stability particularly at high temperatures; otherwise its general physical properties are not detectably altered. Therefore, DNA so labeled can be used for physical and hybridization studies.
INTRODUCTION
Many studies of polynucleotides require that these polymers be radioactive. At present, the most widely used means of obtaining radioactive polynucleotides is the in vivo method of labeling. However, where the organisms are large or rare, or where rates of nucleic acid biosynthesis preclude efficient labeling in an entire organism, one of two alternatives must be used.The alternate most often employed, tissue culture, retains m a n y of the disadvantages of other in vivo procedures. The second alternate is the in vitro labeling of isolated polynucleotides. Most of the documented procedures of in vitro labeling either use excessive amounts of radioactive isotopes 1 or leave the polynucleotides degraded z,3. A methylation procedure has been used for RNA with some degree of success 4 although this method does not seem to be useful for DNA z. Hence, a versatile and economical method that can introduce radioisotopes with minimal alterations of the isolated polymers would be of considerable value. When polynucleotides are treated with sodium borohydride in the presence o~ ultraviolet irradiation, a selective reduction of the pyrimidines occurs 5. The reactivity of the bases, specifically thymine in DNA 6 and uracil in RNA s, suggests a useful method for introducing radioactive isotopes into polynucleotides. This paper discusses the application of the reaction in labeling DNA and RNA and examines the physicochemical properties of the modified DNA product. * S u b m i t t e d in p a r t i a l fulfillment of t h e r e q u i r e m e n t s for a M a s t e r of Science Degree from t h e D e p a r t m e n t of B i o c h e m i s t r y U n i v e r s i t y of W a s h i n g t o n , Seattle, W a s h . 98105, U.S.A.
Bioehim. Biophys. Acta, 238 ~t97 I) 174-.179
LABELING OF POLYNUCLEOTIDESBY PHOTOREDUCTION
175
MATERIALS DNA's from sea urchin and Bacillus subtilis were the gift of Mrs. Margaret Farquhar of this department. Calf thymus DNA was obtained from Miles Chemical Co. B. subtilis E14ClDNA was the gift of Dr. Mary-Dell Chilton of this department. NaB3H4 was obtained from Amersham-Searle Corp. Hydroxylapatite was obtained from Clarkson Chemical Co. Bovine serum albumin was obtained from Armour Pharmaceutical Co. All other chemical compounds were standard ACS reagent grade. Buffer was o.15 M NaCl-o.oI5 M sodium citrate, pH7.2. An ultraviolet lamp, UVS-I2, was obtained from Ultraviolet Products, Inc., San Gabriel, Calif., U.S.A.
METHODS AND RESULTS
Photoreduction o/ polynucleotides A typical in vitro labeling experiment is the following: A sample of native DNA ( o . I - 0 . 5 m g / m l ) o r RNA (0.5 mg/ml) was dissolved in o.o165 M sodium borate buffer, p H 9.0. Nitrogen gas was bubbled through the solution to displace oxygen which might interfere with the reaction. An aliquot was then transferred to a quartz cuvette and gently stirred throughout the reaction. A 5- to Io-fold molar excess of NaB3HI (relative to DNA thymine) freshly dissolved in the borate buffer was added to the solution of polynucleotides. This solution was immediately placed behind a i - c m quartz cuvette containing 20 % acetic acid solution which served to filter ultraviolet light below 2400 A. The lamp charactelistics were such that a distance of 17.8 cm from the reaction cuvette gave a dosage of 347 ° ergs/mm 2 per rain at 253.7 nm. After 15 min of irradiation the reaction was stopped by the addition of o.I ml of o.I M sodium citrate, p H 5.3, for every I.O ml of nucleic acid. The excess NaBSHI decomposed with vigorous bubbling. The nucleic acids were then either directly dialyzed versus 0.05 M sodium citrate, p H 5.3, or alcohol precipitated prior to dialysis. After 4 or 5 changes of dialysis buffer when the radioactivity of the dialysate was reduced to background level, the nucleic acids were transferred to o.15 M NaC1O.Ol5 M sodium citrate buffer and subsequently stored at --20 ° in the same buffer. All photoreductions were run in the fume hood. The concentrations of the labeled nucleic acids were measured on a Cary 15 spectrophotometer, and their radioactivities measured in the Beckman CPM-Ioo liquid scintillation system.
Labeling o/ di//erent polynucleotide preparations under various conditions Experiments were performed to test the effects of certain reaction parameters on the extent of labeling. The concentrations of all DNA solutions were 0.595 mg/ml in o.o165 M borate buffer, p H 9.0. For denaturation, the samples were placed in boiling water for IO min. The NaBSH4 stock solution was 0.276 M in the alkaline borate buffer, and this solution was used within I rain after it was made. Identical aliquots containing 2.76/zmoles wele added to all the nucleic acid solutions for photoleduction. All samples were irradiated simultaneously. Irradiation periods were o, 2, 5, IO, 15, 30 and 60 rain for native DNA, and IO and 30 min for denatured DNA. For one sample of DNA that received no irradiation, reduction was allowed to pro-
Biochim. Biophys. Acta, 238 (i97 r) 174-179
176
v.F.
LEE, M. P. GORDON
ceed for 3o min. The RNA solution was I mg/ml and the irradiation time was 15 rain. The results of these experiments are shown in Fig. I.
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Fig. I. 8H i n c o r p o r a t i o n as a f u n c t i o n of irradiation t i m e . All D N A r e d u c t i o n s were r u n a t a ratio of i m M t h y m i n e to 5.3 m M NaBSH4. T h e R N A r e d u c t i o n w a s r u n at a ratio of 0.89 m M uracil to 5.3 mY[ NaBSH4. (~)-O, n a t i v e D N A , p h o t o r e d u c e d ; /x, n a t i v e D N A , r e d u c e d w i t h o u t i r r a d i a t i o n ; IN, d e n a t u r e d D N A , p h o t o r e d u c e d ; × , R N A , p h o t o r e d u c e d . Fig. 2. Sucrose g r a d i e n t s e d i m e n t a t i o n of calf t h y m u s D N A . iN- - -El, unlabeled, u n t r e a t e d D N A , a b s o r b a n c e p a t t e r n ; O - O , i n vitro labeled D N A , a b s o r b a n c e p a t t e r n ; A - & , in vitro labeled D N A , radioactivity pattern.
Band sedimentation o/the labeled DNA Neutral sucrose gradient sedimentation. Photoreduced calf thymus DNA (irradiated 15 rain), o.500 mg per 0. 4 ml, was layered on a 4.6 ml linear sucrose gradient, 6-3 ° %. The gradient was centrifuged at 48 ooo rev./min (approx. 2 o o o o o × g ) for 75 min at 15 ° in the SW 5o.1 rotor in the Beckman L2-65 B centrifuge. Io-drop fractions were then collected and their absorbances measured at 260 nm; 0.8 ml of distilled water was added to each fraction. The fractions were then dialyzed against three changes of O.Ol5 M NaCl-o.ooI 5 M sodium citrate buffer. In order to determine the 3H content of each fraction, 0.2 ml bovine serum albumin (I mg/ml) was added followed b y 1.2 ml of a 50 % trichloroacetic acid solution. After IO min, the solutions were filtered through W h a t m a n GF/C filters which were then dried and counted in IO ml of toluene-liquiflor scintillation fluid. A sample of untreated calf thymus DN A was also subjected to band sedimentation under the same conditions. Fig. 2 shows the patterns obtained in this experiment. Alkaline sucrose gradient sedimentation. A mixture of equal amounts of unlabeled and labeled calf thymus DNA (irradiated 15 min), 12o/~g total in 0. 4 ml solution, was layered on a 4.6-ml linear alkaline sucrose gradient (6-30 % sucrose in 0. 7 M NaCl-o.3 M NaOH). The tubes were then centrifuged at 49 ooo rev./min (approx. 225 ooo ×g) fol 5 h at 25 ° in the SW 5o.1 rotor in the Beckman L2-65B centrifuge. At the end of the centrifugation, Io-drop fractions were again collected and treated as above. The data from this experiment are shown in Fig. 3. Heat stability o! the labeled DNA Capillaries containing 5 °/~1 aliquots of sheared labeled B. subtilis DNA (2oo Biochim. Biophys, Acta, 238 (1971) i 7 4 - I 7 9
ABELING OF POLYNUCLEOTIDES BY PHOTOREDUCTION
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Time {hours)
Fig. 3. Alkaline sucrose gradient sedimentation of calf thymus DNA. The gradient contained cold and labeled material in the ratio of i : I. O - Q ) , absorbance pattern; &-&, radioactivity pattern. Fig. 4. Heat stabilities of labeled B. subtilis DNA. - - , specific activities of in vitro labeled DNA; - - -, specific activities of in vivo labeled [x4C]DNA. The horizontal lines indicate samples which had not been heated. Q), denatured DNA, incubated at 60°; A native DNA, incubated at 70°; El, native DNA, incubated at 80°; 0 , native DNA, incubated at 9o°.
/zg/ml, 13o c o u n t s / m i n per #g, in o.15 IV[NaCl-o.oI5 M sodium citrate buffer) were sealed a n d t r e a t e d in the following m a n n e r : For the 60 ° heat s t a b i l i t y study, the capillaries were kept at IOO° for IO min before being transferred to a 60 ° b a t h a n d i n c u b a t e d for as long as 23 h. I n the heat s t a b i l i t y studies carried out at 7 o°, 80 °, a n d 9 °0 , the samples were i n c u b a t e d for different lengths of time w i t h o u t prior denaturation. All samples were r u n in duplicate. A t the end of the i n c u b a t i o n periods, the capillaries were opened a n d their contents diluted i n 6 m l of 0.5 M phosphate buffer, p H 7.2. The r a d i o a c t i v i t y of the trichloroacetic acid-precipitable m a t e r i a l was t h e n determined. Samples of sheared 14C-labeled B. subtilis D N A were t r e a t e d identically. Fig. 4 shows the heat stabilities of the various samples. M e l t i n g curves o / t h e labeled D N A Melting curves of cold a n d labeled D N A were o b t a i n e d s i m u l t a n e o u s l y using the Gilford 20o0 spectrophotometer. All m e a s u r e m e n t s were made in o.15 M NAC1o.o15 M sodium citrate buffei. The Tm's a n d hyperchromicities at 260 n m are given in Table I.
TABLEI MELTING
C U R V E D A T A F R O M in
DNA
LABELED
NaBSH4 Irradiation specific activity time (min) (mC/mg)
Sea urchin
-
-
3.7 157.o Calf thymus
oitro
-
-
3.7 3.7
DNA DNA T,. specific activity (counts/rain per /~g)
Hyperchromicity at 260 nm
-15 io
o 260 87oo
83.8 83.3 83. 3
o.261 o.268 o.268
-IO 60
o 203 191
85.5 85.2 85. 3
0.345 0.350 0.363
Biochim. Biophys. Acta, 238 (i97 I) 174-179
178
V . F . LEE, M. P. GORDON
Renaturation kinetics o/ the labeled D N A as measured on hydroxylapatitd Photoreduced B. subtilis DNA, specific activity 13o counts/min per #g, was dissolved in o.15 M NaCl-o.oI 5 M sodium citrate buffer at a concentration of 200 #g/ ml or co = 0.606" lO -3 moles nucleotide per l. The solutions were then sheared in a French pressure cell at IO ooo-12 ooo lb/inch 2. Aliquots of 5oF1 were sealed in capillary micropipettes. The samples were denatured at ioo ° for IO min, then quickly transferred to a 60 ° bath. At intervals, samples were removed for measurement of renaturation by hydroxylapatite column chromatography. A reference sample of in vivo labeled B. subtilis DNA was treated identically. For this control sample, the initial co was 1.52- lO -3 moles nucleotide per 1. The results are shown in Fig. 5.
........
i
'
....
,"1
,
, ,,,'"1
,
, ......
i
I00
90~ 8O
~, 6o ~ 5o
~ 4o ~o 20
I0
\ \\
C)--(~) io vivo l a b e l e d ~ In vitro Iobeled O.I
~
I
Got (molt
\\\
tO x
I00
lee/liter)
Fig. 5. R e n a t u r a t i o n curves of B. subtiEs D N A measured by h y d r o x y l a p a t i t e c h r o m a t o g r a p h y .
DISCUSSION
The application of the photoreduction reaction to polynucleotides is promising in its convenience, its economy, and its relative safety. The alterations in the DNA produced by ultraviolet irradiation and incorporation of 3H produce minimal changes in the physical-chemical properties. It was found that the optimum concentration of native DNA was about 0. 5 mg/ml with a 5- to Io-fold molar excess of NaBSH4 relative to the thymine content. Higher concentrations of native DNA solution resulted in lower reaction rates probably due to the increased viscosity. The use of sheared or denatured DNA at higher concentrations would bypass the problem of viscosity. The results in Fig. i indicate that incorporation reaches a plateau after IO mill. Since photoreduction is known to proceed to ring cleavage, and in view of ultraviolet-induced lesions, most photoreactions were allowed to proceed only lO-15 min. It was estimated that one out of sixty thymine moieties had been altered, assuming one 3H substituent per base modified. Adjusting this value to the 58 % ( A + T ) content of calf thymus DNA s, the number of base pairs altered is o.97 °/o. This must be considered an upper limit since it is possible that there is more than one 3H per reduced base. Denatured DNA gave 25-35 % higher incorporation under the same conditions. RNA also showed comparable incorporation. Since some 3H was also incorporated without irradiation Biochim. Biophys. Acta, 238 (1971) 174-179
LABELING OF POLYNUCLEOTIDES BY PHOTOREDUCTION
179
(see Fig. I), a second reaction other than photoreduction could be occurring simultaneously. The sucrose gradient band sedimentation of the labeled DNA was a clear indication that the radioactivity was incorporated throughout the DNA (see Fig. 2). However, the alkaline sucrose gradient patterns (Fig. 3) of the absorbance and radioactivity suggest that minor degradation of the DNA has occurred. Compared to the in vivo labeled E14C]DNA, the photoreduced samples showed lowered thermal stabilities at high temperatures. However, the stability at 60 ° indicates that the photoreduced DNA is useful for a variety of hybridization studies. A 1. 5 % mismatching of bases results in a lowering of the T m by I ° (ref. 9)If the lowered T,,'s (Table I) are truly indicative of base mispairing and not due to technical limitations, an upper limit of 0.8 % of base mismatching is indicated. This number is in agreement with the calculations obtained for the number of base pairs altered, 0.97 %. The widths of the melting transitions were found to be essentially unchanged in the photoreduced samples. As measured by hydroxylapatite chromatography, the renaturation properties of the in vitro and in vivo labeled DNA's are identical (Fig. 6). A published value of cot½ = 5 for B. subtilis 1° was measured by absorption spectroscopy. Renaturation rates are usually 2-fold faster when measured by the hydroxylapatite method as compared to the optical method since the former measures the fraction of fragments reassociated, while the latter measures the total strand length reassociated 9. Allowing for this, our value of Cot~ = 3 compares favorably with the adjusted value of 2.5 for B. subtilis, measured under similar salt and temperature conditions. These results are in agreement with the stability of [3H]DNA at 60 °. It was recently brought to our attention that Dr. L. Kirkegaard and coworkers at the University of Wisconsin have prepared ZH-labeled tRNA, mRNA and rRNA b y procedures similar to those described here n.
ACKNOWLEDGMENTS
It is a pleasure to thank Dr. Brian J. MCCARTHY for his interest and helpfulness. This work was supported by funds from the U.S. Atomic Energy Commission, the National Science Foundation, and the Graduate School Research Fund, University of Washington. REFERENCES I 2 3 4 5 6 7 8 9 Io II
E. BORENFREUND, M. S. ROSENKRA~IZ AND A. BENDICH, J. Mol. Biol., i (1959) 195. F. ASCOLI AND F. IV[. KAHAN, J. Biol. Chem., 24I (1966) 428. D. G. SEARCH, Biochim. Biophys. Acta, 166 (1968) 36o. K. D. SMITH, J. L. ARMSTRONG AND B. J. McCARTHY, Biochim. Biophys. Acta, 142 (1967) 323. P- CERUTTI, K. IKEDA AND B. WITKOP, J. Am. Chem. Soc., 87 (1965) 2505. C-. BALLE, P. CERUTTI AND B. WITKOP, J. Am. Chem. Sot., 88 (1966) 3946. G. BERNARDI, Nature, 2o6 (1965) 779. C. LONG, Biochemists' Handbooh, D. V a n N o s t r a n d Co., P r i n c e t o n , N.J., 1961, p. 199. R. J. BRITTEN AND D. KOHNE, Science, 161 (1968) 529. B. L. McCoNAUGHY, C. D. LAIRD AND B. J. McCARTHY, Biochemistry, 8 (I969) 3289. L. KIRKEGAARD, P h . D. Thesis, U n i v e r s i t y of Wisconsin, 1969. C o m m u n i c a t e d b y Dr. R. M. BOCK, U n i v e r s i t y of W i s c o n s i n , Madison, ~risc.
Biochim. Biophys. Acta, 238 (1971) 174-179