Tue JournaL or Biotogican CHEMISTRY Vol. 238, No. 9, September 1963 Printed in U.S.A. Chemically Synthesized Deoxy polynucleotides as Templates for Ribonucleic Acid Polymerase* Arturo Fanascut,t Junius ADLER, anp H. G. Kuorana From the Departments of Biochemistry and Genetics and the Institute for Enzyme Research, the University of Wisconsin, Madison 6, Wisconsin (Received for publication, April 25, 1963) The enzymatic synthesis of ribonucleie acid from the four ribonucleoside 5’-triphosphates in the presence of deoxyribo- nucleic acid has been documented in a number of laboratories (1-11). The reaction is catalyzed by an enzyme called RNA polymerase and the DNA determines the composition and the sequence of nucleotides in the synthesized RNA according to the base-pairing principle first recognized for DNA by Watson and Crick (7-11). The RNA synthesized in this manner stimu- lates the incorporation of amino acids into protein (12-15). A deoxypolythymidylate fraction obtained by chemical syn- thesis (16) was tested previously by Hurwitz et al, (7, 11) and was found to bring about the synthesis of ribopolyadenylic acid. In the present work, purified synthetic deoxypolynucleotides of known size and sequence have been used to study further the mechanism of action of RNA polymerase. The results reported herein show that deoxyoligonucleotides as small ag the penta- nucleotide dT; can serve as templates for the synthesis of ribo- polyadenylate.' The rate of the synthetic reaction increases with an increase in the size of the deoxypolythymidylate until a chain length of 14 is reached, and this polymer was actually more active than DNA. The product synthesized from deoxy- polythymidylate of various sizes was invariably very much larger than the size of the template. Several lines of evidence showed that the enzyme initiated the synthesis of new chains, rather than causing esterification to the 3’-hydroxyl end of the deoxy- polynucleotide chains. Finally, in these simpler systems, the incorporation of the ribonucleoside 5/-triphosphates again fol- Jowed the Watson-Crick base-pairing principle, although some exceptions were noted. A brief report of these findings has already appeared (17). * Supported by grants from the National Institutes of Health, the National Science Foundation, Life Insurance Medical Re- search Fund, and the Graduate School of the University of Wis- consin. { Fellow of the International Laboratory of Genetics and Bio- physics, Naples, Italy. Present address, Department of Bio- chemistry, Stanford University Medical School, Palo Alto, Cali- fornia, ? All of the chemically synthesized polynucleotides used in the present work carry a 3'-hydroxyl group at one end and a 5’-phos- phate group at the opposite end. For convenience, this class of homologous polynucleotides is simply designated in the text by the nucleoside initial with a subscript indicating the number of nucleosides in the chain. For example, deoxy-pTpTpTpTpT is dTs and deoxy-pCpCpCpCpCpC is dCs, ete. A, G, C, U, and T stand for adenosine, guanosine, cytidine, uridine, and thymidine. EXPERIMENTAL PROCEDURE RNA polymerase was purified from Escherichia coli according to the procedure described by Chamberlin and Berg (10). #. cold phosphodiesterase (18) was a gift from Dr. I. R. Lehman. C™- Ribonucleoside 5’-triphosphates were purchased from Schwara BioResearch, Inc. Deoxyribopolynucleotides—Deoxy cytidine, deoxyadenosine, and deoxyguanosine oligonucleotides were prepared by published procedures (19-21). The homologous deoxythymidine poly- nucleotides dT, to dT, were prepared as described previously (16, 22), and CJabeled thymidine polynucleotides were pre- pared by Mr. W. J. Connors by adaptation (23) of these pub- lished procedures (16, 22). Pure thymidine polynucleotides dTy. to dTy, and a fraction containing members higher than dTy4 were prepared from the 1 M triethylammonium bicarbonate fraction obtained in an experi- ment described previously (Khorana and Vizsolyi, Table I, and Fig. I (16)). The latter fraction, as mentioned earlier (16), was a complex mixture of polynucleotides containing apparently a high proportion of oligonucleotides linked together by pyrophos- phate bridges between the terminal 5’-phosphomonoester groups, with general structure pT (pT) .pT “4 \ pT (pT) pT The mixture (110 optical density units at 267 my) was treated in dry pyridine (2 ml) with 0.5 ml of acetic anhydride for 3 days in order to selectively cleave the pyrophosphate bonds, and the reaction mixture was worked up as described earlier (22). Chro- matography on a DEAE-cellulose (carbonate form) column (50 X 1.2 em) gave a series of peaks which were processed by the standard method developed earlier (16). As a result of cleavage of the pyrophosphate bonds, more than 50% of the ultraviolet- absorbing material was present as a mixture of oligonucleotides smaller than dTj. The higher homologues were then applied to paper along with previously characterized dT,; as marker. The dTy», dT,3, and dT traveled with progressively decreasing Hp values when chromatographed for 2 weeks in descending n-propy! alcohol-concentrated ammonia-water (55:10:35). Acetylation of 3'-Hydrozyl End Groups in Penta- and Undeca- thymidylic Acids—An aqueous solution of ammonium salt of the polynucleotide (2 zmoles of thymidine) was passed through a column (1 X 2 em) of Dowex 50 ion exchange resin (H+) and 3080 September 1963 the total effluent and washings evaporated after addition of pyridine (1 ml). To the residue was added triethylamine (0.05 ml) and pyridine (2 ml) and the solution was re-evaporated with vacuum from an oil pump. The residue was rendered anhydrous by repetition of evaporation after addition of dry pyridine. Finally, dry pyridine (0.5 ml) and acetic anhydride (0.2 ml) were added and the sealed reaction mixture kept in the dark at room temperature for 4 hours. Water (2 ml) was then added and the total solution kept for 2 hours at room temperature. It was then evaporated under reduced pressure to an oil which was extracted with dry ethyl ether several times. The insoluble polynucleotide material was obtained as a fine solid deposit on the wall of the round bottom flask. It was dissolved in water and the aqueous solution was lyophilized. The solid residue was made up to 0.2 ml with water. Resistance of Polynucleotides Bearing Terminal 3'-O0-Acetyl Groups to E. coli Phosphodiesterase—An exonuclease purified from E. colt has been shown by Lehman (18) to degrade deoxy- ribopolynucleotides in a stepwise manner from the end bearing a, 3’-hydroxyl group. The reaction produces deoxyribonucleo- side 5’-phosphates until the chain length is reduced to the dinucleotide (18). In a control experiment, 0.17 pmole of the tetranucleotide d-pTpTpTpT was incubated at 37° in a 0.2-ml incubation mixture in the presence of 0.02 ml of 1 m Tris buffer (pH 7.5), 0.02 ml of 0.1 m magnesium chloride, and 100 units (18) of enzyme. Degradation to d-pT and d-pTpT was com- plete in under 2 hours as determined by paper chromatography of aliquots in descending ethy] alcohol-0.5 m ammonium acetate buffer, pH 3.8 (7:3, v/v). Incubation of the 3’-O-acetyl d- pTpTpTpTpT under identical conditions up to 4 hours showed complete resistance of the oligonucleotide to the enzyme. Ina second experiment the use of a 3-fold higher concentration of the enzyme preparation under the above conditions showed like- wise the absence of any degradation. On the other hand, the degradation of oligonucleotides bearing 3’-hydroxyl groups pro- ceeded normally in the presence of the 3'-O-acetyl derivatives, showing that the latter were not inhibitory. Assay of RNA Polymerase—The reaction mixture was exactly the same as that described by Chamberlin and Berg (10), except that synthetic deoxypolynucleotides usually replaced DNA. When DNA was used as primer, the C4“ RNA synthesized was measured after precipitation with perchloric acid, exactly as described by Chamberlin and Berg (10). When synthetic deoxypolynucleotides were used, .a new assay was devised in order to be able to detect any low molecular weight ribopolynucleotides that might be too small to precipitate in acid. The reaction was stopped with 0.02 ml of concentrated ammonium hydroxide and the entire mixture was then deposited in one pipetting onto the origin of a 2.5- x 57-cm strip of DEAE- cellulose? paper (Whatman DE-20). Descending chromatog- raphy was carried out in 0.8 mM ammonium formate for 23 hours. Under these conditions polynucleotides as small as ribotetra- adenylate remain at the origin, whereas unused nucleoside tri- phosphates move away. The strip was then left to dry in air. The area containing the C™-ribopolynucleotides (from 2.5 em below to 2.5 cm above the origin) was cut out, folded in half at the origin, placed folded up in a scintillation vial containing sol- vent (3 g of 2,5-diphenyloxazole and 100 mg of 1,4-bis-2-(5- phenyloxazolyl)benzene per liter of reagent grade toluene), and 2 The form DEAE- refers to diethylaminoethyl-. A. Falaschi, J. Adler, and H. G. Khorana 3081 counted at 2-80 (875 volts) and window settings of 10, 50, and 100 in a Packard Tri-Carb liquid scintillation spectrometer. Determination of Size of Ribopelynucleotides—After the area containing the C'-ribopolynucleotide had been counted as de- scribed above, it was cut into small pieces and incubated in 2 mi of 0.8 Nn NaOH for 20 hours at 37° to hydrolyze the ribopoly- nucleotide. The liquid was then filtered and chilled in ice. It was neutralized by adding slowly small amounts of dry Dowex 50 (H*) resin; after each addition, about 5 minutes with occasional stirring were allowed before the pH was measured. When the pH reached 7, 5 wl of 1 nN NaOH were added in order to avoid any dephosphorylation which could occur if the pH became less than 7 during the subsequent manipulations. The supernatant liquid, combined with 0.1 N ammonium hydroxide washings of the resin, was concentrated by lyophilization and spotted on a strip, 2.6 em X 57 em, of DEAE-cellulose paper. Descending chromatography in 0.2 m ammonium formate was carried out for 6 hours until the front reached the end of the paper. This served to separate added (as markers) adenosine, adenosine 3’-phosphate, and adenosine 2’(3’) ,5’-diphosphate. The dried strip was cut at 1-cm intervals and these pieces were put into scintillation vials and counted as described above. The ratio of radioactivity in adenylic acid to adenosine or in adenylic acid to the adenosine diphosphate was considered the average size of the ribopolynucleotide. RESULTS Deoxypolythymidylate as Template for Synthesis of Ribepolyadenylate Effect of Chain Length on Rate of Synthesis—The initial rate of incorporation of adenylate varied with the size of the deoxypoly- thymidylate at saturating concentrations of each polymer, as shown in Fig. 1. With dT; no activity was observed under the conditions used, and with dT, there was occasional activity. But dT; always brought about significant, although small, incorporation of adenylate. With further increase in size the effectiveness of the polymers then rose, at first slowly up to dT7, then with a big leap upward between lengths of 8 and 11. The activity reached ° ¢ nN + PORATED/IOMIN. 10) mMOLES INCOR 4 6 8 190 l2 4 16 CHAIN LENGTH Fic. 1, Effect of chain length on the activity of deoxypoly- thymidylate. The experimental procedure for measuring the incorporation of adenylate from Cl-ATP is described under “Assay of RNA Polymerase.”’ 3082 Tasie I Efficiency of DNA and deoxypolythymidylate in stimulating RNA polymerase The experimental procedure is described under “Assay of RNA Polymerase.” Incorporation of nucleotide Polymer Only C'4-ATP UTP, and present CATE present mumoles/10 min Calf thymus DNA................... 14.1 0.82 Heated calf thymus DNA*........... 4.8 3.05 Deoxypolythymidylate, 1 m fraction. / 20.0 AD ace e ees | 34.0 *Thymus DNA was heated by immersion in a boiling water bath for 10 minutes, then chilled in ice. a maximum at dT, and dTi;. Table I shows that dTy, is actu- ally more active for ribopolyadenylate synthesis than DNA is for the synthesis of RNA in the presence of all four nucleoside triphosphates. Hurwitz et al. (7, 11) had already shown that the 1 M triethyl- ammonium bicarbonate fraction referred to above is active for the synthesis of ribopolyadenylate. We have now confirmed the activity of the same 1 m fraction and have found it to be about 60% as active as dTy, (Table I). This lesser activity is probably due to the presence of less active polymers or inhibitors in the mixture. As mentioned above, the 1 m fraction contains polymers of various sizes larger than dTy, and also oligonucleo- tides linked together by pyrophosphate bridges. It seems very likely that such pyrophosphate compounds are inhibitors for the priming action of polynucleotides. RNA polymerase will catalyze the synthesis of ribopoly- adenylate when DNA is present together with ATP as the only nucleoside triphosphate (10). Denatured DNA is a preferred primer for this activity (24).3 Table I shows a confirmation of these results and a comparison of DNA with the activity of the 1 m fraction and dTy,. For the synthesis of ribopolyadenylate dT, is the most active polymer. In order to be sure that the comparison of the results with polynucleotides of different sizes was meaningful, it was neces- sary to check that the primer did not undergo degradation during the incubation with RNA polymerase (see also below). C4-Labeled dTz was used in the usual reaction mixture except that ATP was omitted. The mixture was incubated as usual and then put on a DEAE-cellulose carbonate column (0.6 x 27 cm) and cluted with a linear gradient of triethylammonium bicarbonate from 0 to 0.48 m (total volume, 500 ml). More than 99.7% of the radioactivity appeared as a single peak in the posi- tion corresponding to dT;. This ruled out the possibility of detectable breakdown of the polymer by the enzyme preparation. Effect of Chain Length on Saturating Concentration of Tem- plate—A saturating concentration was determined for each poly- mer that stimulated the synthesis of ribopolyadenylate in the experiment of Fig. 1. For dT;, dT, and dT, the results are plotted in Fig. 2 according to the method that Lineweaver and Burk have used for substrates (25). The polymer concentra- tions which gave half-maximal rates with dT7, dTu, and dTi.4 3M. Chamberlin and P. Berg, unpublished observations. Synthetic Deoxypolynucleotides as Templates for RNA Polymerase Vol. 238, No. 9 were found to be 50 X 1076, 20 x 1078, and 2.0 x 107° m, re- spectively. Increasing the size of the polymer not only increases the maximal velocity of the reaction (as shown also by Fig. 1) but decreases very strikingly the concentration of polymer re- quired for half-saturation. Apparently the affinity of the deoxy- polythymidylate for the enzyme increases markedly with size be- tween dTy and dT. Size of Ribopotyadenylate Formed—The C'-ribopolyadenylate formed in the presence of deoxypolythymidylate of various sizes was hydrolyzed with sodium hydroxide to C-adenosine, C'adenosine 2'(3’)-phosphate, and a C-material that resem- bled adenosine 3’,5’-diphosphate but was not further char- acterized. The details are described under “Experimental Pro- cedure.” The ratio of radioactivity in adenylic acid to adenosine and in adenylic acid to the adenosine diphosphate was taken to be the average size of the ribopolyadenylate. Table IT lists the results for the size of the products formed from dT;z, dTs, dT», dTu, and dTy4; for smaller thymidine oligonucleotides too little product was available to provide significant results. The estimate of the chain length of the products is only approximate, owing to the inaccuracy of counting on DEAE paper the small amounts of radioactivity in the adenosine and in the adenosine diphosphate region. The most striking feature of the results is that the product is of a much larger size than the deoxypoly- thymidylate. No marked differences are apparent between the sizes of the products obtained with different sized deoxypoly- thymidylates. Evidence for Noninvolvement of Terminal 8'-Hydroxyl Groups of Polythymidylate in Ribopolyadenylate Formation—During chromatography of the total alkaline hydrolysate of the ribopoly- adenylate (see above), no significant amount of radioactivity ‘aT 7 A 1S Lob 0.5- dT) 005 010 O15 020 025 15 B dT AOL LL dy v 14 05 1 ! l 1 05 os 5 20 25 Cc Fic. 2. Reeiprocal plot of the effect of concentration of deoxy- polythymidylate on the incorporation of adenylate. A compares dT; and dTu; B compares dT and dT. The concentration of polymer is expressed as millimicromoles of polymer (not nue- leotide equivalents) per ml; v, millimicromoles of adenylate incorporated from ATP per 10 minutes, as measured by the DEAE-cellulose paper assay described under ‘‘Assay of RNA Polymerase.” September 1963 remained at the origin of the chromatogram where the deoxyribo- polythymidylate remains adsorbed. This result indicated that ribopolyadenylate synthesis was not initiated by esterification of the terminal 3’-hydroxyl group in deoxypolythymidylate to form a phosphodiester linkage. Such a linkage would have been resistant to alkaline hydrolysis and a product of the general structure (Diagram I) would have resulted. T T T A OH \ P P P P. P Diagram I To test further whether the 3’-hydroxyl group is essential in the synthesis of ribopolynucleotides, 3’-O-acetyl-dT,, was pre- pared for testing as a primer. Table III (Lines 1 and 3) shows that 3’-O-acetyl-dT,; is about as active as dT. The concen- tration of 3’-O-acetyl-dT, required to half-saturate the enzyme was very similar to the concentration already found for dT. This diminished the possibility that a small amount of unacety- lated dT}, was the active component in the 3’-O-acetyl-dT 1, prep- aration. Furthermore, 3’-O-acetylthymidylate was found to be Tas ie II Size of ribopolyadenylate formed from deoxypolythymidylate tenvplates The experimental procedure is described under ‘‘Size of Ribo- polyadenylate Formed.” Radioactivity Chain length Polymer Ad : ; ; diphosphate Adenyiy | Adenosine | Bya | B/C | Average c.p.m, aT, 29 2,550 96 73 27 50 dTs 80 10,100 135 125 74 100 dT, 70 4,680 32 67 146 107 dT 1 34 3,590 86 101 46 7 dT 14 114 9,080 63 79 144 111 TaBLe III Acetylated deoxypolythymidylate as template for RNA polymerase Treatment with #. coli phosphodiesterase was carried out at 37° in a 0.15-ml reaction mixture containing 100 units (18) of enzyme, 0.02 ml of 1 m Tris buffer (pH 7.5), 0.01 ml of 0.1 m mag- nesium chloride, and 0.4 umole of nucleotide equivalent of poly- nucleotide. Then 0.02-ml aliquots of this were added to an RNA polymerase reaction mixture without prior inactivation of the diesterase, since it could be shown that the presence of diesterase does not interfere with the synthesis of ribopolyadenylate. Polymer Treatment with | Incorporation of phosphodiesterase ; adenylate from ATP | mumoles/30 min dT LLe eee eee _ | 2.30 C6 + 0.04 3'-O-acetyl-dTir... 200.00. ..0.00. - | 2.37 3/-O-acetyl-dTi......-...000--. + 1.32 A. Falascht, J. Adler, and H. G. Khorana 3083 TasLe IV Deoxypolycytidylate as template for synthesis of rtbopolyguanylate The experimental procedure is deseribed under “Assay of RNA Polymerase.”’ Incorporation Incorporation of Polymer of guanylate from GTP adenylate from ATP myumole/10 min myumoles/10 min dCs <0.05 dCs <0.05 dCs <0.05 dC, 0.15 dCio 0.17 dT, 3.2 stable when incubated with the RNA polymerase preparation; this served to exclude any acylase activity. In order to ensure that none of the unacetylated dT, was present, the preparation of the acetylated polynucleotide was preincubated with the #. coli phosphodiesterase. Acetylation of the 3’-hydroxyl group in thymidine oligonucleotides confers resistance toward this enzyme (see above). Table III (Lines 2 and 4) compares dT), and 3'-O-acetyl-dTy; after treatment with phosphodiesterase. This enzyme abolishes nearly all of the activity of dT\, whereas most of the activity of 3’-O-acetyl-dTy; remains resistant to phosphodiesterase. It may be concluded that 3’-O-acetyl-dT,; is active for the synthesis of ribopolyadenylate, and that a free 3’-hydroxyl group of deoxypolythymidylate is not essential for ribopolyadenylate synthesis to oceur. It follows that addition of adenylate to the 3/-hydroxyl end of deoxypolythymidylate is not a necessary part of ribopolyadenylate synthesis. Deoxypolycytidylate as Template for Synthesis of Ribopolyguanylate Incubation of deoxypolycytidylate with RNA polymerase and GTP led to the formation of ribopolyguanylate, and the rate of the reaction depended on the size of the deoxypolycytidylate (Table IV). Whereas a chain length of 5, 6, or 8 proved too small to be effective, dC, and dC, showed significant activity. The dependence of the reaction on the concentration of dC Is shown in Fig. 3; half-saturation occurred at 5.4 x 10-8 a. A comparison of the deoxypolycytidy late series with the deoxypoly- thymidylate series shows that lower homologues are more effec- tive in the latter series (Table IV) but a lower concentration of the deoxypolycytidylate is required for saturating the enzyme. Deoxypolycytidylate of much higher molecular weight than used here, prepared from the deoxypolycytidylate-deoxypolyguanylate polymer (26), has been shown by Chamberlin and Berg to be active for the synthesis of ribopolyguanylate (27). Experiments with Deoxypolyguanylate and Deoxypolyadenylate In the deoxypolyguanylate series, only dGg was tested. It proved to be inactive for the incorporation of nucleotide from CTP. This may be explained by the finding that even small homologues of deoxypolyguanylate have a high tendency to form aggregates of very large molecular weight (28). Chamber- lin and Berg (27) have reported that deoxypolyguanylate pre- pared from the deoxypolycytidylate-deoxypolyguanylate poly- mer is also inactive for the incorporation of cytidylate. 3084 40 3.0- I/V 20+ oO | al 0.2 0.3 I/CONCENTRATION OF POLYMER Fie. 3. Reciprocal plot of the effect of concentration of dCi) on the incorporation of guanylate. The experimental procedure is described under ‘‘Assay of RNA Polymerase.’’ The concentra- tion of polymer is expressed as millimicromoles of polymer (not nucleotide equivalents) per ml; v, millimicromoles of guanylate incorporated from GTP in 10 minutes, as measured by the DEAE- cellulose paper assay described under ‘Assay of RNA Polymer- ase,’’ TaBLe V Specificity of incorporation of nucleotide The experimental procedure is described under ‘‘Assay of RNA Polymerase.”’ Nucleotide incorporated Polymer ATP only GTP only | CTP only |ure only | mumoles/10 min Poly dT, 1 m fraction 8.7 |<0. 05-0.65/ <0.05 | <0.05 dC io <0.05 0.69 <0.05 | <0.05 Poly dA, 1 m fraction 1.4 <0.05 <0.05 | <0.05 dGe | <0.05 Deoxypolyadenylate homologues of sizes up to dAs and a 1 M fraction that contained a mixture of homologues larger than dAg failed to show significant incorporation of nucleotides from UTP. Possibly, conditions other than those used so far might be effec- tive for the synthesis of ribopolyuridylate. Specificity of Incorporation of Nucleotides In the presence of the 1 m fraction of deoxypolythymidylate, no significant incorporation of CTP or UTP was observed in 10 minutes, under conditions in which 8.7 mmoles of adenylate from ATP were incorporated (Table V, Line 1). Surprisingly, 0.65 mumole of guanylate from GTP was incorporated under the same conditions. As the enzyme preparation became older, the incorporation of guanylate disappeared at a time when the in- corporation of adenylate had decreased by only 60%. Furth, Hurwitz, and Anders in one experiment (7) also noted a small incorporation of guanylate. Deoxypolycytidylate brought about specific incorporation of guanylate (Table V, Line 2). With dC,o, 0.69 mymole of nucleo- tide was incorporated from GTP but there was no significant incorporation of nucleotide from ATP, CTP, or UTP. With the 1 m fraction of deoxypolyadenylate, the incorporation of 1.4 mumoles of nucleotide from ATP was unexpectedly ob- served (Table V, Line 3); in this experiment there was no signifi- cant incorporation of nucleotide from UTP, CTP, or GTP. Synthetic Deoxypolynucleotides as Templates for RNA Polymerase Vol. 238, No. 9 DISCUSSION The present work has demonstrated that short chain deoxy- polynucleotides serve as templates for the synthesis of ribopoly- nucleotides in the presence of RNA polymerase. The effective- ness of the homologous members in the reaction increases with an increase in chain length, the maximal rate in the case of the polythymidylate series being reached with dT. It is note- worthy that the maximal rate here obtained was higher than the rate normally obtained when all the four ribonucleoside triphos- phates and DNA are used. The saturating concentrations of the thymidine polynucleotides decreased with an increase in chain length and the enzyme showed high affinity even for the short chain length dTy. A major point of interest established by the present work has been that the ribopolynucleotide synthesis does not begin by adding nucleotides to the terminal 3’-hydroxyl group of the deoxyribonucleotide. This conclusion is based primarily on two findings: alkaline hydrolysis of the ribopolyadenylate product leaves no detectable adenylate in the deoxypolythymidylate, and dT carrying a 3’-O-acetyl group is still effective in the synthesis of ribopolyadenylate. Throughout this paper we have referred to the deoxypolynucleotides as ‘‘templates” for RNA polymerase rather than “primers” because the synthesis of the product in- volves the formation of new complementary chains instead of elongation of chains. Since addition to the end of the deoxypoly- nucleotide is not necessary, it may be that replication can begin anywhere along the template; this has significance at the bio- logical level, for it allows the synthesis of messenger RNA from any one of the genes in a DNA molecule without requiring syn- thesis from all. Although in the present work the Watson-Crick type of base- pairing was ordinarily observed, the slight incorporation of deoxy- guanylate in the presence of thymidine polynucleotides was noted, an observation which has also been made previously (7). A further noteworthy exception, which merits further study, was the formation of ribopolyadenylate when deoxyribopolyadenyl- ates were used as templates. The reaction resembles the previ- ously documented ribopolyadenylate-primed synthesis of ribo- polyadenylate (29), and its biological significance remains unknown. Under the conditions tested no polyuridylate syn- thesis occurred when deoxypolyadenylate was used as template. The failure is perhaps due to the lack of appropriate conditions, since the polyriboadenylate-dependent polyuridylate synthesis has already been found to be very sensitive to temperature and to the presence of a critical concentration of manganous ions (30). RNA polymerase brings about the synthesis of ribopoly- adenylate when DNA is present and ATP is the only substrate (10, 24). The reaction proceeds best when denatured DNA is used (24). The size of the ribopolyadenylate formed has been estimated to be in the range of 60 to 70 (10) and 400 (24). To explain that the expected short runs of polythymidylates in DNA could bring about the synthesis of much larger sized ribopoly- adenylate, Chamberlin and Berg (10) postulated a “slippage” mechanism whereby a run of AMP residues would slip along the sequence of thymidylate residues, leading to an elongation of the polyadenylate chain. The present work indicates that it would take a run of only five to seven thymidylates in DNA in order to synthesize ribopolyadenylate of a much larger size. All evidence points to the involvement of a single enzyme for the DNA-dependent synthesis of RNA and of ribopolyadenylate September 1963 (24) Since the synthesis of ribopolynucleotides observed here is so similar to the DNA-dependent polyriboadenylate synthesis, and since the same purified enzyme (10) was used here, it seems most probable that the syntheses studied in this work are due actually to RNA polymerase rather than some contaminating enzyme. The assay used in the present work involved separation of the product from the unreacted labeled nucleoside triphosphate by anion exchange chromatography on a DEAE-cellulose paper. Oligonucleotides as short as the tetranucleotide could be sepa- rated from the nucleoside triphosphates under the conditions used. This assay was developed to detect low molecular weight polynucleotides, which could be missed by the usual assay (10) that depends on acid insolubility of polynucleotides. The assay may be generally applicable in studies on polynucleotides, for example in determining the initial events in DNA and RNA synthesis. A related method based on the use of DEAE-cellulose paper has been previously reported by Bollum (31). The ribopolynucleotides obtained by the use of chemically synthesized deoxypolynucleotides and RNA polymerase may be expected to bring about the specific incorporation of amino acid into protein when added to an amino acid-incorporating system (32, 33). For example, the ribopolyadenylate synthesized here would be expected to lead to incorporation of lysine (33). SUMMARY Short chain thymidine polynucleotides serve as templates for the synthesis of ribopolyadenylate in the presence of RNA poly- merase. The effectiveness as template increases markedly with size. Thus, thymidine pentanucleotide shows detectable ac- tivity whereas the maximal activity is reached with tetradeca- nucleotide, the latter being more active for ribopolyadenylate synthesis than thymus DNA is for ribopolyadenylate or RNA synthesis. The product formed from the different sized tem- plates has in each case an average chain length of 50 to 100. The synthesis of ribopolyadenylate has been shown not to involve addition to the 3’-hydroxyl ends of polythymidylate. Deoxypolycytidylate larger than the octanucleotide brings about the synthesis of ribopolyguanylate. Deoxypolyadenylate has failed so far to give synthesis of polyuridylate. With deoxy- polyadenylate the synthesis of ribopolyadenylate is noted. Acknowledgment—We wish to acknowledge the technical assistance of Miss Rachael Gettle. REFERENCES 1. Weiss, 8. B., anp GiapstonE, L., J. Am. Chem. Soc., 81, 4118 (1959). 2. Wuiss, 8S. B., Proc. Natl. Acad. Sct. U. S., 46, 1020 (1960). 3. Hurwitz, J., BRESLER, A., AND Drrincer, R., Biochem. and Biophys. Research Communs., 3, 15 (1960). A. Falaschi, J. Adler, and H. G. Khorana 3085 4. Stevens, A., Biochem. and Biophys. Research Communs., 3, 92 (1960); J. Biol. Chem., 236, PC43 (1961). 5. Burma, D. P., Kroaur, H., Ocnoa, S., WarNmR, R. C., AND Writt, J. D., Proc. Natl. Acad. Sct. U.S., 47, 749 (1961). 6. Furts, J. J.,. Hurwitz, J.. anp ANvERs, M., J. Biol. Chem., 237, 2611 (1962). 7. Furtu, J. J., Hurwrrz, J., anp Goupman, M., Biochem. and Biophys. Research Communs., 4, 362 (1961). 8. Weiss, S. B., anp Nakamoto, T., Proc. Natl. Acad. Sct. U.S., 47, 1400 (1961). 9, Huane, R. C., ManesHwari, N., anp Bonner, J., Biochem. and Biophys. Research Communs., 3, 689 (1960). 10. CHamBEeRLIN, M., AND Bere, P., Proc. Natl. Acad. Sct. U. S., 48, 81 (1962). 1l. Hurwirz, J., Furra, J. J., ANpERs, M., ano Evans, A., J. Biol. Chem., 237, 3752 (1962). 12. Woop, W., anp Buna, P., Proc. Natl. Acad. Sci. U.S., 48, 94 (1962). 13. Nina, C., aND Stevens, A., J. Molecular Biol., 6, 650 (1962). 14. Ersensrapt, J. M., Kameyama, T., anp Nove, G. D., Proc. Nail. Acad. Sci. U.S., 48, 659 (1962). 15. Furtn, J. J., Kanan, F. M., ano Hurwitz, J., Biochem. and Biophys. Research Communs., 9, 337 (1962). 16. Kuorana, H. G., anv Vizsouy1, J. P., J. Am. Chem. Soc., 83, 675 (1961). 17. Favascui, A., ADLER, J., AND KHorana, H. G., Federation Proc., 22, 462 (1963). 18. Lenman, I. R., J. Biol. Chem., 235, 1479 (1960). 19. Kaorana, H. G., Turnur, A. F., anp Vizsotyi, J. P., J. Am. Chem. Soc., 88, 686 (1961). 20. Raupn, R. K., anp Kuorana, H.G., J. Am. Chem. Sec., 83, 2926 (1961). 21. Raueu, R. K., Connors, W. J., ScHauver, H., anp KHorana, H.G., J. Am. Chem. Soc., in press. 22. Kuorana, H. G., Vizsoty1, J. P., anb Raupu, R. K., J. Am. Chem. Soc., 84, 414 (1962). 23. Kuorana, H.G., anp Connors, W. J., Biochemical prepara- tions, in press. 24. Stevens, A., Abstracts of papers presented at the American Chemical Society meetings, Atlantic City, New Jersey, September 9-14, 1962, p. 2 C. 25. LinEWEAVER, H., anp Burg, D., J. Am. Chem. Soc., 66, 658 (1934). 26. Rappine, C. M., Jossz, J., anD Kornpena, A., J. Biol. Chem., 237, 2869 (1962). 27. CHAMBERLIN, M., and Brera, P., Federation Proc., 21, 385 (1962). 28. Rauru, R. K., Connors, W. J., anp Kuorana, H.G., J. Am. Chem. Soc., 84, 2265 (1962). 29. Epmonps, M., anp Asprams, R., J. Biol. Chem., 237, 2636 (1962). 30. Weiss, 8. B., Symposium on informational molecules, Rutgers University Press, New Brunswick, New Jersey, 1962, in press. 31. Boutum, F. J., J. Biol. Chem., 287, 1945 (1962). 32. Jones, O. W., JR., AND NrRENBERG, M. W., Prec. Natl. Acad. Sci. U.S., 48, 2115 (1962). 33. GaRDNER, R.S., Wausa, A. J., Basriio, C., Miter, R. S., LENGYEL, P., aND Spryer, J. F., Proc. Natl. Acad. Sct, U.S., 48, 2087 (1962).