Proc. Nat. Acad. Sci. USA Vol. 71, No. 12, pp. 5017-5021, December 1974

Deoxysubstitution in RNA by RNA Polymerase In Vitro: A New Approach to Sequence Determinations (pancreatic DNase I/U1 /U2 ribonuclease/ A/ribosubstituted DNA) GARY V. PADDOCK, HOWARD C. HEINDELL AND WINSTON SALSER Biology Department and Molecular Biology Institute, University of California at Los Angeles, 405 Hilgard Ave., Los Angeles, Calif. 90024 Communicated by Charles Yanofsky, August 12, 1974 ABSTRACT Deoxynucleotides have been incorporated substituted RNA. We have observed that Mn++ ion will not into RNA synthesized in vitro by RNA polymerase with only cause DNA polymerase to synthesize ribosubstituted either double-stranded or single-stranded DNA as a to synthesize template. By use of this technique to block or promote DNA but will also cause RNA polymerase cleavage at a particular phosphodiester bond, a variety of deoxysubstituted RNA. Subsequently we have discovered a specific cleavages may be obtained with the available ribo- number of other papers dealing with the synthesis of deoxy- and I. These methods should substituted RNA (12-14). These papers did not, however, greatly increase the ease and rapidity of nucleotide se- point out the applicability of the approach to nucleotide quence determinations. sequencing and did not include the controls we have found The introduction of radiographic approaches by Sanger and essential for demonstrating that complete deoxysubstitution his colleagues (1-4) greatly increased the power of nucleotide has occurred. sequencing techniques, but there remain certain obstacles In this preliminary paper we discuss the theory by which whose solution could result in impressive further increases in substitution of deoxynucleotides becomes an aid in nucleotide the rapidity with which large sequences may be determined. sequencing, the technical precautions necessary to eliminate Examples of such problems, some merely irritating and others traces of contaminating ribonucleotides that would otherwise major, range from difficulties in ordering long tracts of pyr- be preferentially incorporated, and the evidence that the imidines to difficulties in obtaining good yields and purities of system preserves fidelity adequate for nucleotide sequencing the partial digestion products needed to obtain the order of studies. T1 or pancreatic RNase digestion products. In the case of ordinary RNA sequencing techniques the MATERIALS AND METHODS most serious difficulties spring from the lack of base-specific cleavages at other than G which are needed to U1 RNase, DNase free, was the kind of gift of C. A. Dekker obtain overlaps. An improvement in this situation can be (15, 16), U2 RNase and T1 RNase were purchased from Cal- achieved by using ribosubstitution DNA sequencing tech- biochem. Spleen , pancreatic RNase A, and niques (5-9) in which one ribonucleotide is substituted for electrophoretically purified DNase I were purchased from one of the four deoxynucleotides during in vitro synthesis Worthington Biochemical Corp. This DNase I contained with DNA polymerase I. The product can then be degraded, RNase activity that was deactivated by treatment with so- cleaving at every ribose linkage by using alkaline hydrolysis dium iodoacetate (17). Hemoglobin (Hb) mRNA was sup- or the appropriate ribonuclease digestion. Unfortunately the plied by A. Bank and hemoglobin complementary DNA oligonucleotides resulting from such ribosubstitution cleavages (cDNA) was prepared and supplied by D. Kacian (18-20). cannot themselves be further cleaved at a specific base (in Dipodomys ordii (kangaroo rat) HS-,3 satellite DNA was pre- theory this could be achieved by synthesizing DNA sub- pared and supplied by J. Mazrimas and F. Hatch (21). Bac- stituted with two ribo-bases, such as G and C, but in fact the teriophage M13 DNA that had been depurinated and hy- rate of DNA synthesis is reduced to intolerably low levels drolyzed to give fragments of about 10 S was the gift of Kirk when this is attempted). Consequently such oligonucleotides Fry. must be sequenced using digestions with micrococcal nu- RNA polymerase prepared by the method of Burgess (22) clease (7, 9) or DNase I (unpublished work of B. Wallace in was a gift from C. Climenson and D. Eisenberg. a-82P- this lab) or by partial digestions with spleen or venom phos- labeled-nucleoside triphosphates were purchased from New phodiesterases (10, 11). England Nuclear Corp. and were purified of unidentified Ideally, one would like not only to be able to cleave spe- RNA polymerase inhibitors on a charcoal column (9). Non- cifically at several individual bases (as can be done by making radioactive nucleoside triphosphates were purchased from several ribosubstituted , each with a different ribo- Calbiochem and Sigma Chem. Co. We have found that it is base), but also to be able to cleave the same labeled product at extremely important to purify deoxynucleotides of contaminat- several different specific bases in successive digestions. This ing ribonucleotides, and for this purpose the method of Wu could be accomplished if it were possible to synthesize deoxy- (23) has proved convenient and effective. This is followed by purification on a DEAE-cellulose column with elution by tri- Abbreviations: cDNA, DNA complementary to RNA; Hb, ethylammonium bicarbonate, pH 8.5, in a 0.01 M-0.5 M gradi- hemoglobin. ent and subsequent desalting by passage through a Bio-Gel P2 5017 Downloaded by guest on September 25, 2021 5018 Biochemistry: Paddock et al. Proc. Nat. Acad. Sci. USA 71 (197 )

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n of ,,paw. 9L.. a 9. 1A 1B 3A 3B 3C FIG. 1. Autoradiographs of two-dimensional fingerprints of Hb RNA. [a-32P]UTP-labeled Hb RNA (Fig. 1A, far left) and [a-32P]- UTP-labeled dC-substituted Hb RNA (Fig. 1B, middle left) were digested with U1 RNase and bacterial alkaline and the products were separated by electrophoresis in two dimensions by the techniques described in Sanger et al. (1). The oligonucleotides are labeled as in Poon et al. (24). B is the blue dye marker and P is the pink dye marker. The origin is denoted by X. FIG. 3. Aatoradiographs of two-dimensional fingerprints of Hb RNA. [a.32P]CTP-labeled Hb RNA (Fig. 3A, middle); [a-32P]CTP- labeled, dT-substituted Hb RNA (Fig. 3B, middle right); and [a-32P]dCTP-labeled Hb RNA (Fig. 3C, far right) were digested with pancreatic RNase A and the products were separated by electrophoresis in two dimensions by the techniques described in Sanger et al. (1). 2':3'-Cyclic phosphates are denoted by > Oligonucleotides in Fig. 3C with the mobilities of U-Up, U-Up, and U-U-Up may have arisen from incomplete digestion of poly(U) by pancreatic RNase A. The poly(U) was labeled by [a-32P]rUTP contaminant.

column. The 32P-labeled deoxynucleotides obtained from New were present. The deoxysubstituted RNA was subjected to England Nuclear Corp. were used without further treatment. hydrolysis overnight at 370 with 0.2 N LiOH and the diges- Synthesis of 32P-labeled Hb RNA was carried out as in ref. tion products were separated by electrophoresis on DEAE 24. Synthesis of deoxysubstituted RNA was carried out in paper at pH 3.5 or with 7% formic acid buffer. Each of the 100 Mul of 0.1 M Tris * HCl, pH 7.5, 2.5 mM MnCl2 containing digests was found to contain ribo-mononucleotides plus approximately 10 ,ug of RNA polymerase, 1 ,ug of DNA, 20 larger fragments. For example, when deoxy-A-substituted nmol of each ribonucleoside triphosphate, and 100 nmol of RNA was synthesized using [a-32P]GTP, radioactivity was the selected deoxynucleoside triphosphate except in the case found not only in Cp*, Gp*, and Up* but also in dAp*Gp, of the nucleoside triphosphate carrying the radioactive label. dApdAp*Gp, as well as lesser amounts of label in dApCp* 32P-Labeled nucleoside triphosphate (1 mCi) with a specific and dApdApCp* and dApUp*. Higher homologues (with activity of approximately 100 Ci/mmol was used per syn- more dA residues) also appeared to be present but were not thesis. The reaction mixture was made 0.2 M in KCl when further characterized. These results were confirmed in sep- double-stranded HS-3 DNA was the template. Incubation arate experiments in which the radioactivity was introduced was for 45 min at 37°. The product was then desalted and on the deoxynucleoside triphosphate. purified of nucleoside triphosphate precursors by passage of deoxysubstitution synthesis through a Bio-Gel P-60 column. (See Note Added in Proof.) Fidelity Nucleotide sequencing techniques and ribonuclease diges- These results suggested that it would be possible to achieve tions were those described in Sanger et al. (1), Barrell (2), the desired specifications of cleavage by using the deoxysub- Fry et al. (7), and Whitcome et al. (9). Digestion with pan- stitution technique. For example, we hoped that RNA deoxy- creatic DNase I was with 10 of 1.25 mg/ml of in substituted at C residues could be cleaved specifically at U by 0.1 M Tris HCl, pH 7.0, 0.02 M MgCl2, 2 mM CaCl2 over- treatment with pancreatic RNase or specifically at dC by night at 370 with 0l/ug of carrier RNA added. treatment with DNase I. Regardless of the apparent facility with which deoxysubstituted RNAs can be specifically RESULTS cleaved, the technique would be useless unless the fidelity of Incorporation of deoxynucleotides synthesis under deoxysubstitution conditions is good so that In our initial experiments we measured the ability of Esche- the correct sequences are obtained. In order to test the fi- richia coli RNA polymerase to catalyze incorporation of delity we have compared the sequences of normal RNA and a-32P-labeled nucleoside triphosphates into acid-precipitable deoxysubstituted RNA synthesized from two well-charac- material when one of the ribonucleoside triphosphates was re- terized DNA templates; the HS-/3 satellite of Dipodomys placed by the corresponding deoxynucleoside triphosphate. ordii (7), and Hb cDNA (24). Single-stranded M13 DNA was used as template and in- corporation of radioactive label was found to occur in all * The asterisk is used to indicate the position of the labeled 32P cases at a level comparable to when all four ribonucleotides atom. Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Deoxysubstitution in RNA by RNA Polymerase In Vitro 5019

One of the most revealing comparisons may be obtained by cleaving both normal and deoxysubstituted RNAs at the same base (or bases) and directly comparing their finger- prints. For instance, Figs. 1A and B show a comparison of U-Jp RNA labeled with [a-82P]UTP and deoxy-C-substituted up u.-p ..,,.. .*.Z

RNA labeled with [a-'2PIUTPt. As can be seen, the two up ... *s fingerprints are identical and the fingerprint of the dC-sub- stituted material showed normal relative yields of all spots, including those with significant runs of dC, such as spots 7 (dC-dC-dC-U-G), 26 (dC-dC-U-dC-dC-dC-U-G), and 29 i iz (dC-dC-dC-dC-U-U-G). cup This experiment provides a rather stringent test of the fidelity of the deoxysubstitution technique. Possible mistakes are of two types, random and systematic. By random errors we mean general "sloppiness" of the enzyme, in which it Cp% *4 might make, for example, an error 10% of the time at each AADH ~ ~ P.i' base. Errors of this kind would result in greatly reduced yields of the major spots ("correct" sequences) and the ap- pearance of numerous heretofore absent and generally un- reproducible minor spots. The fingerprints we have ob- CPI~ b c d |a b~ tained are "clean" (i.e., devoid of numerous minor spots ex- cept in those cases where the deoxynucleotide was contam- FIG. 2. Pancreatic RNase A digestions of oligonucleotides inated with ribonucleotides) and provide good evidence obtained from RNase Ul + phosphatase digestions of [a_!32P]_ against this sort of mistake. dCTP-labeled Hb RNA were carried out as in Barrell (3) with By systematic errors we mean that the RNA polymerase an enzyme (E) to substrate (S) ratio of 1:10 (w:w) and the sequences consistently, as for products were separated on DEAE paper at pH 3.5. In addition, might make particular wrong and 7 instance if a region coding for the fragment A-U-U-C-U-C-G for oligonucleotides 4 (dC-dC-U-GoH) (dC-dC-dC-U- GOH), the following were carried out: (a) no reaction, (b) alkaline consistently yielded A-U-U-U-U-dC-G rather than A-U-U- hydrolysis with 0.2 N LiOH, (c) pancreatic RNase digest E:S = dC-U-dC-G under deoxy-C-substitution conditions. Any 1:10 (w:w), and (d) pancreatic RNase digest E:S = 1:100 mistakes of this sort could be very easily detected by com- (w: w). The other oligonucleotides digested with pancreatic paring Figs. 1A and B, since they would result in easily RNase are identified as follows: 26, dC-dC-U-dC-dC-dC-U-GoH; recognizable changes in position of the major spots, some- 29, dC-dC-dC-dC-U-U-GoH; 31, U-dC-dC-dC-A-dC-U-Goz; 30, thing that we have not observed. dC-dC-U-U-dC-A-GoH; 22, dC-U-dC-dC-U-Goa; 28, A-U-dC- dC-U-GoH; 33, dC-dC-U-A-U-dC-A-Goz; 40, U-U-dC-dC-U-GoH; Enzyme cleavage characteristics 42, U-dC-dC-U-dC-U-GoH; 44, A-dC-U-U-dC-U-Goi. All Cs in The sequences of the fragments shown in Fig. 1A are known the figure are to be understood to be dCs. Unidentified tracks in from the work of Poon et al. (24). Consequently ribonuclease the figure show digestions of mixtures or of oligonucleotides whose T1 fingerprint patterns such as that shown Fig. 1B provided sequences are not yet determined. B and P are the blue and pink us with many pure deoxy-C-substituted fragments of known dye markers, respectively. sequence on which we could test the cleavage techniques that we desire to use for further sequence determinations. For ex- same products, dCp*dCpUp and dCp*dCp*dCpUp, respec- ample, aliquots of such fragments were digested with pan- tively, as digestion with pancreatic RNase. Fragment 26, creatic RNase A (which would normally cleave at both C and which could not be definitively sequenced with conventional U residues, but in this case only cleaves at U residues) and the methods, yielded dCp*dCpUp* and dCp*dCp*dCpUp upon resulting fragments were further analyzed after separation by digestion with pancreatic RNase. Complete spleen digestion electrophoresis on DEAE paper at pH 3.5. The analysis of of these two products showed that dCp*dCpUp* yielded some of the larger fragments in the T1 RNase fingerprint is label in both UMP and CMP, indicating that dCp*dCpUp*- not yet complete but all of the available evidence suggests dCp*dCp*dCpUpG is the correct sequence. In a similar G that the expected radioactive fragments were obtained in label experiment the sequence of fragment 26 was confirmed, each case. since the only pancreatic RNase product labeled was dCpd- In Fig. 2 we show the digestion with pancreatic RNase A CpdCpUp*. of fragments taken from an experiment similar to that in Fig. Similar experiments with dT-substituted [a-_2P]CTP- 1B except that the label was introduced as [a-32P]dCTP. labeled RNA shows that cleavage products ending in C may Digestion of fragment 4, whose sequence is dCp*dCpUpGOH, be obtained by digestion with pancreatic RNase. For ex- yielded dCp*dCpUp; fragment 7, with sequence dCp*dCp*- ample, Ul-phosphatase product ApdTp*Cp*CpdTpG yielded dCpUpGoH yielded dCp*dCp*dCpUp; fragment 22, with se- ApdTp*Cp* as the only labeled pancreatic RNase digestion quence dCpUp*dCp*dCpUpG yielded dCpUp* and dCp*- product. dCpUp, as shown in Fig. 2. In the case of spots 4 and 7, com- New fingerprint cleavage patterns yielded the plete hydrolysis with LiOH was performed and One of the major new advantages of a deoxysubstitution t U1 RNase was used instead of Ti RNase to obtain cleavages RNA sequencing scheme is that one can obtain specific pri- after G in deoxysubstituted RNA because U1 RNase is known mary cleavages at U residues (by dCTP substitution and to be DNase free (personal communication from A. Blank). cleavage with pancreatic RNase A), or at C residues (dTTP Downloaded by guest on September 25, 2021 5020 Biochemistry: Paddock et at. Proc. Nat. Acad. Sci. USA 71 (1974) substitution, cleavage by pancreatic RNase A), or at A res- picted in Fig. 3B showed evidence of rUTP contamination idues (dGTP substitution, cleavage by U2 RNase). even though the dTTP had been periodate treated. Perhaps Comparison of the fingerprint resulting from pancreatic rUTP was present in the commercial rCTP as a deamination RNase A cleavage of RNA (Fig. 3A) with the dT-RNA or product. If so, chromatography of the rCTP or other pre- dC-RNA (Figs. 3B and C) shows the expected differences. In cursors should solve the problem. It should be pointed out the fingerprint of dT-substituted RNA (Fig. 3B), the normal that such problems are accentuated under our conditions of pancreatic RNase products ending with U are missing (ex- limiting Hb cDNA template where the total incorporation is cept for small amounts of U due to some contaminating only 5-7%. When we use larger quantities of more plentiful UTP present in this particular experiment). Similarly the templates, such as phage M13 DNA, 20-30% of the radio- usual pancreatic RNase fragments ending in C are missing active precursor is incorporated into product and the relative in the fingerprint pattern of dC-substituted RNA (Fig. 3C). amounts of rU contamination in a dT-substituted product Also many new spots, not observed in Fig. 3A, are seen in were greatly reduced. Figs. 3B and C. Although we have only begun the task of Another phenomenon which we have observed is escape sequencing each of these new fragments in Figs. 3B and C, it synthesis similar to that discussed by Salser (26) for ribosub- is already obvious that most of them are the expected new stitution reactions. It is observed that if RNA polymerase has products containing internal dT residues (Fig. 3B) or internal an alternative to incorporating dNTPs, it will incorporate the dC residues (Fig. 3C). corresponding rNTP preferentially. For example, our Hb In addition cleavage of [a-32P]CTP-labeled dG-substi- cDNA was primed for its synthesis by reverse transcriptase tuted Hb RNA with RNase U2 (which cuts at rA and rG from Hb mRNA with oligo(dT). In our normal RNA syn- residues) gives the fingerprint pattern (not shown) that we thesis, if [a-32P]rATP is used we obtain 90% of the label in would expect to result from an A cleavage. poly(A), the poly(A) being made from the oligo(dT) as a template. Similarly if [a-32P]rUTP is used about 10% of the Cleavage by pancreatic DNase I label is found in poly(U) [the poly(U) is probably made from We were hopeful that DNase I could be used to cleave spe- the poly(A) as a template]. However, if dC-RNA is labeled cifically at deoxynucleotides in deoxysubstituted RNA. Pre- with [a-32P]UTP, then about 30% of the label is in poly(U), liminary results suggest that DNase I can digest large deoxy- and if dG-RNA is labeled with [a-32P]UTP, then 90-100% of substituted RNA molecules but not small (5 to 7 nucleotide) the label is in poly(U). We have not attempted synthesis of fragments containing deoxynucleotides. This may be related deoxysubstituted Hb RNA with A label, but we suspect the to the well-known fact that DNase I works more rapidly on product would be entirely poly(A). Our results suggest that large molecules than on small molecules even when the sub- these problems are peculiar to Hb cDNA or other molecules strate is entirely DNA (autoretardation-see ref. 25) or to the with stretches of oligo(dT) at their 5' terminus. One can take fact-that the enzyme has no ability to cleave di- or trinucleo- advantage of this situation with appropriate experimental tide fragments even with DNA substrates. design. An experiment with [a-32PldTTP-substituted Hb The ability of DNase I to degrade large deoxysubstituted RNA gave a "clean" Ul-phosphatase fingerprint [no poly DNAs may provide a method for producing large "partial" (dT) on the origin], and one can guess that [a-32P]dATP, if digestion fragments in good yield. It should also be noted used, might suppress the normal synthesis of poly(A) that that the small deoxynucleotide containing fragments are occurs with this template. Experiments with M13 single- slowly digested by DNase I that has been treated with iodo- stranded DNA indicate homopolymer synthesis is not a acetate to inactivate contaminating RNase A. But in this problem with this template. case the products obtained are the same when analogous dC- We have also found, in our experiments with HS-,3 double- substituted or non-substituted RNA fragments are digested. stranded DNA as a template, that if one of the four nucleo- Thus this slow digestion is not deoxynucleotide-specific and is tides is predominant in one strand and one deoxysubstitutes presumably due to contaminating iodoacetate-insensitive with that nucleotide, then synthesis of the other strand will or RNase activity rather than the DNase itself. be highly favored. Our experiments suggest that higher levels of deoxynucleo- Synthesis of deoxysubstituted RNA side triphosphates than the corresponding ribonucleoside tri- The only serious problems we have encountered so far are the phosphates are required to achieve maximal synthesis. Sup- result of incomplete deoxysubstitution of the product due to plying the dNTP at a level five times higher than normal in- ribonucleotide contamination of the product (i.e., rC instead creased the yield of the deoxysubstituted RNA by a factor of of dC, for example) causing incomplete blocking of enzyme about three. However, if it is desired to incorporate the 82p cleavages. In early experiments in which the commercial label in the deoxynucleotide, a reasonable level of product deoxynucleoside triphosphates were not repurified we found yield can still be achieved without increasing the concentra- that RNA polymerase exhibited a strong preference for the tion of the deoxynucleotide. ribonucleotide contaminants. Thus in some cases where there was perhaps 1-2% ribose contamination, we observed 30- DISCUSSION 50% ribose contamination in the products. To rid the deoxy- The results depicted in this paper provide several very at- nucleotides of ribose impurities we found that destruction of tractive alternative methods for the determination of nucleo- the ribonucleotides with periodate (23) as described in tide sequences. Because of the variety of specific cleavages Materials and Methods proved to be quite satisfactory. that are possible with deoxysubstituted RNA, a larger set of In some cases particular ribonucleoside triphosphates may primary cleavage products become available, with resultant have to be repurified to remove other contaminating ribo- increased chance of nucleotide sequence overlaps. In small nucleoside triphosphates. For example, the experiment de- molecules, at least, this may negate the necessity of grouping Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Deoxysubstitution in RNA by RNA Polymerase In Vitro 5021

primary digestion products with partial diges- We thank our many colleagues who have supplied us with tions. Furthermore, due to the larger set of options available, gifts of the various DNA templates and used in this work. We are most appreciative for discussions with K. Fry it should be much easier to determine the sequence of these involving DNA sequencing techniques, for the participation by primary digestion products. For example, with an oligonu- D. Browne, R. Main, and R. Friedman in some of the experiments cleotide obtained by RNase U1 digestion to cleave at G res- as part of an undergraduate honors course, and for the technical idues, one has the additional options of dT substitution or assistance of Mrs. S. Chu and Mrs. M. Childs. This research was dC substitution (see Fig. 2) and with supported by U.S. Public Health Service Grants GM-18586 and cleavage pancreatic CA-15940. W.S. was supported by Career Development Award RNase. This can resolve many difficult problems. In addi- USPHS Grant GM-70045. G.V.P. is a Celeste Durand Rogers tion, with dT-RNA, whether it is cleaved at C (pancreatic Cancer Research Fellow. or cleaved at G RNase cleavage) (Ul RNase cleavage), one 1. Sanger, F., Brownlee, G. G. & Barrell, B. G. (1965) J. Mol. has the option of separating any secondary digestion prod- Biol. 13, 373-398. ucts by electrophoresing on DEAE paper in 7% formic acid 2. Brownlee, G. G. & Sanger, F. (1969) Eur. J. Biochem. 11, instead of in pH 3.5 buffer. This procedure yields immediately 395-399. the G+T content merely by inspection of the autoradio- 3. Barrell, B. G. (1971) in Procedures in Nucleic Acid Research, eds. Cantoni, G. L. & Davies, D. R. (Harper and Row, graph. New York), Vol. 2, pp. 751-779. Molecules greater than a few hundred nucleotides in length 4. Brownlee, G. G. & Sanger, F. (1967) J. Mol. Biol. 23, 337- may be too large to be uniquely sequenced using the overlaps 353. provided by primary digestion products. In this case, deoxy- 5. Salser, W., Fry, K., Brunk, C. & Poon, R. (1972) Proc. Nat. substitution of RNA an link between Acad. Sci. USA 69, 238-242. provides important 6. Salser, W., Fry, K., Wesley, R. D. & Simpson, L. (1973) data obtainable from RNA sequencing methods and that Biochim. Biophys. Acta 319, 277-280. available from DNA sequencing techniques. Any of the pri- 7. Fry, K., Poon, R., Whitcome, P., Idriss, J., Salser, W., mary cleavages obtainable with ribosubstituted DNA, from Mazrimas, J. & Hatch, F. (1973) Proc. Nat. Acad. Sci. USA which one can obtain T4 endonuclease IV digestion frag- 70, 2642-2646. 8. Salser, W., Poon, R., Whitcome, P. & Fry, K. (1973) ments (unpublished results of N. Fedoroff in this laboratory), in Proceedings of the 1973 ICN-UCLA Symposium of Molec- can be obtained with deoxysubstituted RNA. Thus with ular Biology, eds. Fox, C. F. & Robinson, W. S. (Academic DNA sequencing techniques one may obtain groupings of the Press, New York), pp. 545-572. primary cleavage products and properly identify them while 9. Whitcome, P., Fry, K. & Salser, W. (1974) in Methods in Enzymology, eds. Grossman, L. & Moldave, K. (Academic the actual determination of their sequences is done through Press, New York), Vol. 29 pp. 295-321. the easier RNA sequencing techniques with the added ad- 10. Ziff, E. B., Sedat, J. W. & Galibert, F. (1973) Nature New vantage of deoxysubstitution. Biol. 241, 34-37. 11. Ling, V. (1972) J. Mol. Biol. 64, 87-102. Note Added in Proof. A preliminary report of these results 12. Cheng, T. Y. & Tso, P. 0. P. (1965) Fed. Proc. 24, 602. was presented on June 3, 1974 as part of the Symposium on 13. Chamberlin, M. (1966) in Procedures in Nucleic Acid Eukaryotic DNA and Structure, FASEB meetings, Research, eds. Cantoni, G. L. & Davies, D. R. (Harper and Minneapolis. Row, New York), Vol. 1, pp. 513-519. After submission of this manuscript we became aware of similar 14. Hurwitz, J., Yarbrough, L. & Wickner, S. (1972) Biochem. Biophys. Res. Commun. 48, 628-635. results of Van de Vorde et al., which have recently appeared else- 15. Glitz, D. G. & Dekker, C. A. (1964) Biochemistry 3, 1399- where (27). 1409. Like these authors we have tested a range of buffer conditions, 16. Kenny, W. C. & Dekker, C. A. (1971) Biochemistry 10, but preliminary results obtained by J. Isaacson in this laboratory 4962-4970. indicate that, with our test template (phage M13 DNA), condi- 17. Zimmerman, S. B. & Sandeen, G. (1966) Anal. Biochem. 14, tions for optimal synthesis seem to be rather different from those 269-277. found by Van de Vorde et al. With dC or dG incorporation 18. Ross, J., Aviv, H., Scolnick, E. & Leder, P. (1972) Proc. we find that added Mg++ in the range 0.5-2.5 mM (while Mn++ Nat. Acad. Sci. USA 69, 264-268. is held constant at 2.5 mM) causes a progressive inhibition of 19. Verma, I. M., Temple, G. F., Fan, H. & Baltimore, D. (1972) Nature New Biol. 235, 163-167. synthesis, and that substitution of Mg++ for Mn++ reduces 20. Kacian, D. L., Spiegelman, S., Bank, A., Terada, M., Meta- synthesis 10-fold. We have observed small stimulatory effects of fora, S., Dow, L. & Marks, P. A. (1972) Nature New Biol. Mg++ but only when the total divalent cation concentration was 235, 167-169. e held at 2.5 mM, far less than that used by Van de Vorde et al. 21. Hatch, F. T. & Mazrimas, J. A. (1970) Biochim. Biophys. Directly comparing our own conditions with those of Van de Acta 224, 291-294. Vorde et al. for dC incorporation, we find that the rate of synthesis 22. Burgess, R. (1969) J. Biol. Chem. 244, 6160-6167. is more than 2-fold greater under our conditions. Obviously it 23. Wu, R. (1970) J. Mol. Biol. 51, 501-521. should be kept in mind that the optimal conditions may be differ- 24. Poon, R., Paddock, G. V., Heindell, H., Whitcome, P., ent for the used in the two laboratories. Salser, W., Kacian, D., Bank, A., Gambino, R. & Ramirez, templates Preliminary F. (1974) Proc. Nat. Acad. Sci. USA 71, 3502-3506. experiments suggest that with other modifications in our condi- 25. Laskowsky, M. (1967) Advan. Enzymol. 29, 165-220. tions (a 2-fold decrease in Tris concentration to 0.05 M, addition 26. Salser, W. (1974) Annu. Rev. Biochem., 43, 923-965. of 0.4 mM dithiothreitol, increase in RNA polymerase concen- 27. Van de Vorde, A., Rogiers, R., Van Herreweghe, J., Van tration to 30,ug/100 ,.d) a 2- to 3-fold further increase in the syn- Heuverswyn, H., Volckaert, G. & Fiers, W. (1974) Nucleic thesis can be obtained. Acids Res. 1, 1059-1067. Downloaded by guest on September 25, 2021