Reprinted from the PRocEEDINGS oF THE NATIONAL ACADEMY OF SCLENCES Vol. 54, No. 2, pp. 579-587. August, 1965. SPECIFIC TEMPLATE REQUIREMENTS OF RNA REPLICASES* By I. Haruna anp S. SpreEGELMAN DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS, URBANA Communicated June 21, 1965 We have previously! reported the isolation of an RNA-dependent RNA polym- erase (termed a “replicase” for brevity) from EH. coli infected with the RNA bac- teriophage MS-2. The purified enzyme showed a mandatory requirement for added RNA and, furthermore, exhibited a unique preference for its homologous. RNA. Ribosomal and sRNA of the host could not substitute as a template and neither of these cellular RNA types showed any ability to interfere with the tem- plate function of the viral RNA. We pointed out! that the ability of the replicase to discriminate solved a crucial problem for an RNA virus attempting to direct its own duplication in an environ- ment replete with other RNA molecules. By producing a polymerase which ignores the mass of pre-existent cellular RNA, a guarantee is provided that replication is focused on the single strand of incoming viral RNA, the ultimate origin of progeny. It seems worth noting that sequence recognition by the enzyme can be of value not only to the virus but also to the investigator. The search for viral RNA rep- licases must perforce be carried out in the midst of a variety of highly active cellular 580 BIOCHEMISTRY: HARUNA AND SPIEGELMAN Proc. N. ALS. polymerases capable of synthesizing polyribonucleotides. If the enzyme finally isolated possesses the appropriate template requirement, a comforting assurance is furnished that the effort expended, and the information obtained, are indeed rele- vant to an understanding of viral replication. Operationally, this view demands that viral RNA be used at all fractionation stages in assaying for polymerase ac- tivity. Our line of reasoning led to the expectation that RNA replicases induced by other RNA viruses would show a similar preference for their homologous templates. An opportunity to test the validity of this prediction came with the isolation of a new and unrelated? # RNA bacteriophage (Qj) in the laboratory of Professor I. Wata- nabe. A modification of the procedure employed to isolate the MS-2 replicase suf- ficed to yield a highly purified and active Qé replicase. It is the primary purpose of the present paper to detail the pertinent properties of the purified enzyme. Comparison of the MS-2 and Q@ enzymes isolated from the same host confirms the predicted requirement. for homology. Each replicase recognizes the RNA genome of its origin and requires it as a template for normal synthetic activity. Materials and Methods.—(1) Bacteria and viruses: The bacterial viruses emploved are MS-2 (originally obtained from Dr. A. J. Clark) and Q¢ (kindly provided by Professor Watanabe). It is essential for any laboratory working with both MS-2 and Q@¢ to monitor continually for contamination of one by the other. Fortunately there is virtually no serological cross reaction between the two,® 4 so that the appropriate antisera can be used for identification and purity checks. The host and assay organism is a mutant Hfr strain of EF. cold (Q-13) isolated in the laboratory of W. Gilbert by Diane Vargo. It has the convenient. property of lacking® ribonuclease T and RNA phosphorylase. Preparation of virus stocks and purified RNA followed the methods of Doi and Spiegelman.? (2) Preparation of infected cells: The basic medium employed for growing infected cells and producing virus contained the following in grams per liter: NIUCI, 1; MgSO.-7 HO, 0.06; gela- tin 1 X 1075; casamino acids (vitamin-free), 15; glycerol, 30; to this is added after separate auto- claving 7 ml of 0.1 M CaCl, and 10 ml containing 4 gm of NagHPO,.-7 H2O0 and 0.9 gm KH2PO,. Lysates in liter quantities are first prepared to be used for infection of larger volumes of cell sus- pensions. These are obtained by infecting log phase cultures (ODe0o of 0.25) with a purified phage preparation at a multiplicity of about 5. They are incubated while shaking at 37°C until lysis is complete and then monitored for titer and purity of the phage. Such lysates can be stored frozen at —17°C indefinitely and thawed just prior to use. In general, 35-liter quantities of cells are grown up in carboys to an ODee of between 0.275 and 0.290. The temperature in the carboys is 34°C, while the temperature of the water bath in which they are immersed is maintained at 37°C. When the cells reach an ODeeo of 0.275, they are infected with virus at a multiplicity of between 10 and 50 and allowed to aerate for mixing for 1 min. The aeration is interrupted for 10 min for absorption, reinstituted, and the incubation continued. At 25 min sufficient sucrose and mag- nesium are added to give final concentrations of 18% and 0.01 M, respectively. After another 5 min the process is terminated by the addition of crushed ice. The cells are harvested in a Sharples centrifuge and stored at —14°C, at which temperature ability to yield active enzyme is retained for periods exceeding 6 months. Uninfected cells are prepared and stored in the same manner. To provide uniform preparations for enzyme isolation, the cells are thawed sometime prior to use and resuspended (20 gm of packed cells in 100 ml) in a solution containing 0.01 M Tris buffer pH 7.4, 0.001 M MgCh, and 0.0005 M mercaptoethanol and 5 ug/ml of DNase. After thorough resuspension with a magnetic stirrer at 4°C, the suspension is divided into convenient aliquots in plastic tubes, frozen, and stored at —14°C. (3) Radieactive substrates: P**-labeled ribonucleoside monophosphates were synthesized as described previously.!. The labeled mononucleotides were converted enzymatically to the corre- sponding ribonucleoside triphosphates by a kinase preparation isolated® from £. colt. (4) Chemical and biological reagents: Unlabeled riboside triphosphates were from P-L Bio- Vou. 54, 1965 BIOCHEMISTRY: HARUNA AND SPLEGELMAN 5S] chemicals, Inc., Milwaukee, Wisconsin. DNase, 2 X recrystallized, was from Worthington Bio- chemical Company, Freehold, New Jersey; it was further purified on DEAE columns to remove contaminating ribonuclease. Phosphoenolpyruvate (PEP) and the corresponding kinase (PEP kinase) were purchased from Calbiochem, Inc., Los Angeles, California; lysozyme from Armour and Company, Kankakee, Illinois; and protamine sulfate from Fli Lilly, Indianapolis, Indiana. The turnip-yellow-mosaic-virus (TYMV-RNA) and the “satellite virus” of the tobacco-necrosis- virus (STNV-RNA) were both kindly provided by Dr. E. Reichmann of the Botany Department at the University of Illinois. (5) Preparation of enzyme: The following procedure is described for 20 gm of packed cells. The frozen cell suspension (120 ml) is thawed and to this is added 0.5 mg/ml of lysozyme, follow- ing which the mixture is frozen and thawed twice, using methanol and dry ice as the freezing mix- ture. To the lysate are added 0.9 ml of 1 M MgCh and 2.5 ug/ml DNase, and the resulting mix- ture is incubated for 10 min in an ice bath. The extract is then centrifuged for 20 min at 30,000 X g and the supernate removed. The pellet is transferred to a prechilled mortar, ground for 5 min, and then resuspended in 30 ml of the same buffer as used for the cell suspension except that the magnesium concentration is raised to 0.01 M to increase the effectiveness of the DNase digestion. The extract is then centrifuged at 30,000 x g for 20 min and the two supernates are combined, adjusted to 0.01 M EDTA (previously brought to pH 7.4), and incubated at 0°C for 5 min. In- soluble proteins appear and are removed by centrifugation at 30,000 X gfor20 min. At this stage, a typical active infected extract has an ODogo of between 150 and 180, Lower values commonly signal a poor infection with a resulting low yield of enzvme. To the cleared supernatant fluid is added 0.01 mg of protamine sulfate for each ODeg unit. After 10 min the precipitate, containing virtually all the enzyme activity, is collected by centrifugation at 12,000 X g for 10 min. It is dissolved in 12 ml of “standard buffer” (0.01 M tris buffer, pl 7.4; 0.005 M MgCl; 0.0005 M mercaptoethanol), adjusted to 0.4 Mf (NH,)sSOu, and allowed to stand overnight at 0°C. This period of waiting is important for the subsequent fractionation since complete disaggregation was found to be essential for acceptable separation of the replicase from transcriptase. The extract is diluted with 24 ml of standard buffer, and after 20 min is centrifuged at 30,000 g for 20 min; for each 40 ml of supernatant are added 12 ml of a 0.5% solution of protamine sulfate. The pre- cipitate which forms contains virtually all of the DNA-dependent RNA polymerase along with an RNA-independent RNA polymerizing activity. The RNA replicase remains in the super- natant and begins to show good dependence on added RNA. [Note: This is one of the critical steps in the fractionation, and any variation in host, medium, time, or temperature of infection modifies the amount. of protamine required to achieve separation. It is often safer to titrate small aliquots and determine the amount of protamine needed by appropriate assays.] After 10 min the precipitate is removed by centrifugation at 12,000 X g for LO min. To the resulting supernate is added an equal volume of saturated ammonium sulfate (saturated at 0°C and adjusted to pH 7.0 with ammonium hydroxide). After 10 min at 0°C the precipitate is collected by centrifugation at 12,000 X g for 10 min and dissolved in 4 ml of standard buffer containing 0.4 Mf ammonium sulfate. The resulting solution is then dialyzed against 1 liter of standard buffer for 1.5 hr. The dialyzed fraction is adjusted (o 0.05 Af ammonium sulfate with standard buffer and passed through a DEAE column (1.2 X 10 ¢m) which is washed with 100 ml of standard buffer just prior to use. After loading the protein, the column is washed with 40 ml of standard buffer containing 0.12 MW NaCl which removes protamine, a poly-A synthetase,’ and residual K-dependent ribonuclease. The enzyme is then eluted with 35 ml of standard buffer containing 0.20 M NaCl. To fractions possessing enzyme activity, saturated ammonium sulfate is added to make the final solution 10% saturation. At this stage, the enzyme preparation has an ODo/ODog ratio of 1.35 and usually contains | mg of protein per ml. Under the ionic conditions specified, no loss in activity is ob- served over a month of storage at. 0°C. (6) The standard assay—assay of enzyme activity by incorporation of radioactive nucleotides: he standard reaction volume ix 0.25 ml and, unless specified differently, contains the following in xmoles: tris HCl pH 7.4, 21; magnesium chloride, 3.2 (when included, manganese chloride, 0.2); CTP, ATP, UTP, and GTP, 0.2 each. The enzyme is usually assayed at a level of 40 yg of pro- tein in the presence of 1 ug of RNA template. The reaction is run for 20 min at 35°C and ter- minated in an ice bath by the addition of 0.15 ml of neutralized saturated pyrophosphate, 0.15 ml of neutralized saturated orthophosphate, and 0.1 ml of 80% trichloroacetic acid. The pre- 582 BIOCHEMISTRY: HARUNA AND SPINKGELMAN Proc. N. A. 8. OD OD SOR 450 ra 40f- _op280 440 oy 30F 430 oy wt 20b x 90260 420 _ Me on 260 \\ ¥. 10F A 410 Xs nN Motte, Ma x Km Mee & 1 oye a wa m 5 10 ay 5 o abd al 6 100 : 100 9 x x x 3 6 9 INFECTED lez @h awa NOT INFECTED w % 4 aw 9 ° 9 ao a ae a SO sO 450 6 oO Qo Zz Zz 7 = a Dua oR ) ° QP. ANA. OR DNA a o we GR NO. TEMPLATE 0 — Temmeare) a ~~ aa | 6 10 1 > «% 1» Fic. 1.—Chromatography on DEAL of second protamine supernatant. Just before collection of fraction no. 1, 35 ml of 0.2 M NaCl in standard buffer was placed on the column, Prior to this, the column had been washed with 0.15 Jf as described in Methods, (5). It will be noted that the peak of enzyme activity is found in the descending shoulder of the OD profile. Rechromatography yields coincidence of the two. cipitate is transferred to a membrane filter and washed seven times with 5 ml of cold 10% TCA. The membrane is then dried and counted in a liquid scintillation counter as described previously.! This washing procedure brings zero ¢ime counts to less than 80 cpm with input counts of 1 x 10° epm. The specific activities of the labeled triphosphates added were adjusted so that with the efficiency employed, 1 X 10° epm corresponds to 0.2 zmoles of the corresponding triphosphate. Results—(A) Properties of the purified QB replicase: Vigure 1 compares the elution profiles of protein and enzyme activity of preparations [see Methods, (5) ] derived from infected and noninfeeted cells. Infected preparations exhibit. a polymerase activity which elutes with 0.2 47 NaCl, responds excellently to added QB-RNA, and is devoid of the DNA-dependent RNA polymerase. The complete dependence on Q3-RNA shows that the enzyme con- tains no nucleic acid which can serve to stimulate the enzyme to activity within the period of assay. A corresponding fraction from uninfected cells shows similar elution properties but possesses no detectable RNA-synthesizing ability. TABLE 1 ASSAY FOR RIBONUCLEASE AND PHOSPHORYLASE C1-ADP incorporation &% FI-RNA Hydrolyzed in 30 Min (30 min), cpm 0.25 M Kt 0.25 M Nat Neither Before DEAE 40* 41 12 <2 After DEAE 32* <2 <2 <2 * Similar preparations from wild type incorporate 30,000 cpm (equivalent to 480 ymoles) before DEAE step and about half that after elution from the column. The assay for phosphorylase was carried out as described by August et al.!! Each reaction (0.25 ml) contained 40 ug protein and 1 zmole of C'4-ADP at 6 X 104 cpm. For the ribonuclease, 10 ug at 1 X 104 epm were added per reaction mixture. VoL. 54, 1965 BIOCHEMISTRY: HARUNA AND SPIEGELMAN 583 TABLE 2 TABLE 3 _Errecr ov Nucumases AND ENeRGY REQUIREMENTS OF Q@ REPLICASE GENERATING System ON QS REPLICASE UMP (or AMP) Cpm . incorporation, Addition Template incorporated Assay mixture yuemoles PEP (1.0 umole) + Complete 390 PEP kmase (10 —QB-RNA 7 ug) Qs 4803 —GTP 5 — 96 —Mg, —Mn 8 DNase (5 ug) Qs 3938 Conditions of assay are those described —_ 45 in Methods, (6), except that Mn++ was RNase (1 pg QB 19 included at 0.2 pmoles per 0.25 ml. —_ 37 None QB 4142 —_— 75 Except for the additions noted, the assays were carried out under the standard conditions described in Methods, (6). Table 1 shows that after the DEAE step the enzyme is completely devoid of ribonuclease I, phosphorylase, and the IC+-stimulated ribonuclease. The presence of the energy-generating system (PEP +- PIP kinase) has little effect on the reac- tion (Table 2), indicating freedom from interfering enzymes which can destroy ri- boside triphosphates. Finally, DNase has no influence on the reaction, whereas the presence of even small amounts of pancreatic ribonuclease completely eliminates the net synthesis of polyribonucleotide. The enzyme system requires not only template but, in addition, all four riboside triphosphates (lable 3). The replicase has an absolute requirement for divalent ions, magnesium being the preferred ion with homologous RNA. Manganese sub- stitutes partially (10%) and induces interesting changes in the nature of the reac- tion, the details of which will be described elsewhere.!” Figure 2 shows the kinetics observed in a reaction mixture containing saturating amounts of template (1 hg RNA per 40 yg of protein). Continued synthesis is observed at 35°C for periods exceeding 5 hr. It will be noted that in 2 hr the amount of RNA synthesized corresponds to 5 times the input template. By variation in the amount of RNA added and the time permitted for synthesis, vir- tually any desired amount of increase of the starting material has been achieved. The cessation of synthesis within 5-10 min reported by others*-!! for presumably similar preparations has been observed by us only in the early stages of purification. Figure 3 examines the effect of added amounts of protein at a fixed level of template (5 pg RNA/O.25 ml). It is evident that the reaction responds linearly, indicating the absence of interfering contaminants in the purified enzyme. (B) Specific template requirements of the replicases: We now turn our attention to the primary question which the present study sought to resolve. Table 4 records the abilities of various RNA molecules to stimulate the Q@ replicase to synthetic activity at the saturation concentration (1 ug) of homologous RNA and twice this level. The response of the Q@ replicase is in aceord with that reported! for the MS-2 replicase, the preference being clearly for its own template. The only heter- ologous RNA showing detectable activity is TY MV and, at the 2-ug level, it supports a synthesis corresponding to 6 per cent of that observed with the homologous Q@- RNA. Both of the heterologous viral RNA’s, MS-2, and STNV are completely inactive and, again, so are the ribosomal and transfer RNA species of the host cell (E.coli Q-13). As might be expected, bulk RNA from infected cells shows some 584 BIOCHEMISTRY: HARUNA AND SPIEGELMAN Proc. N. ALS. ” oO er 45 x 80OF e + o L 5 of OG RNA, Wy 7 x / ta Q fe 6OF +—z___GB- RNA a B © 50 Et g 2° © a E oc 40-+ 2 | 432 9 a n 3.0F 8 ib gs = Zz i > 20Fr @ L 492 a = > O10 NO RNA & | SSL Ke L 4 | 20 40 60 PROT L NO Q@ RNA Ug PROTEIN a Fig. 3.—Response to added protein. As- x ¥ ¥ x says were run for 20 min at 35°C under con- 30 60 90 120 ditions specified in Methods, (6). Again, the incorporation of 4,000 cpm corresponds to the MINUTES ———> synthesis of 1 pg of RNA. Fic. 2.—Kinetics of replicase activity. Each 0.25 ml contained 40 xg of protein and 1 wg of QB-RNA. All other conditions are as specified in Methods, (6). The specific ac- tivity of the UTP was such that the in- corporation of 4,000 cpm corresponds to the synthesis of 1 ug of RNA. templating activity which increases as the infection is allowed to progress. There is no detectable DNA-dependent RNA polymerase activity. To permit a definitive comparison of the two replicases derived from the same host, the MS-2 enzyme was isolated from appropriately infected Q-13. The puri- fication of the MS8-2 replicase followed precisely the same protocol as described for the QS enzyme [Methods, (5) ] except that 0.22 M NaClis used for elution from the DEAE column. The results of the comparison between the two replicases are shown in Table 5 and they are satisfyingly clear-cut. The MS-2 replicase shows no evidence of ac- cepting the Q8-RNA as a template at either level of RNA input. Similarly, the Q6 replicase completely ignores the MS-2 RNA while functioning quite well on its own template. It would appear from these data that the prediction of template specificity is completely confirmed. Discussion.—(1) State of the enzyme and the method of purification: Tt is possible to isolate comparatively pure replicase from suitably infected wild-type Hfr strains.’ However, the use of the mutant Q-13 offered an obvious advantage for the purifica- tion of the replicase since the crude extract was already free of ribonuclease I and phosphorylase. The two remaining interfering activities were due to the DNA- dependent RNA polymerase (transcriptase) and a potassium-stimulated ribonu- clease. It is important to emphasize that the complete removal of the transcriptase is Von. 54, 1965 BIOCHEMISTRY: HARUNA AND SPIEGELMAN {ESPONSE OF QB REPLICASH 'TO DIFFERENT TEMPLATES Template Qs TYMV MS-2 Ribosomal RNA sRNA Bulk RNA from in- fected cells Satellite virus Input Levels of N¢ oo. Acc 1 ug 2 wg 4929 4945 146 312 35 26 45 9 15 57 146 263 61 5] QT x wet TABLE 5 Teme.are Speciricrry or Two RNA REPLICASES -- RNA Templates -—-——-——_. -—MS-2 RNA—~ ——Q8-RN A—— Enzyme 1 pg 2 we 1 pg 2 pe MS8-2 4742 4366 0 56 Qs 36 65 2871 3731 Conditions of assay are those specified in Methods, (6), with Mn** present at 0.2 ~moles per 0.25 ml. DNA (10 pg) 36 Conditions of assay are those specified in Meth- ods, (6). However, as in all cases, assay for DNA- dependent activity is carried out at 10 ne of DNA per 0.25 ml of reaction mixture. Control reactions containing no template yielded an average of 30 epm, essential if questions of mechanism and replicase specificity are to be answered with- out ambiguity. The transcriptase can employ any RNA as templates for RNA synthesis'®: 14 and, in the process, forms a ribonuclease-resistant structure Con- sequently, the use of DNase or actinomycin D does not ensure against confusion with transcriptase activity. In the fractionation procedure described [AMethods, (5)], most of the transcriptase is removed in the precipitate fraction of the second protamine step. The remainder is left behind as a late component in the DEAE column. The potassium-dependent nuclease is tightly bound to cell membrane fragments which are discarded in the low-speed fraction by our comparatively gentle freeze-thaw method of cell rupture. The small amount of ribonuclease that does contaminate the extract is removed from the DEAE column by washing with 0.12 WM NaCl which at the same time eliminates protamine and a poly-A-synthesiz- ing enzyme.? The resulting freedom from interfering and confounding activities makes it possible to study the replicase in a simple mixture containing only the re- quired ions, substrates, and template. (2) Comparison with other viral-induced enzymes: We may summarize the distinc- tive properties of the purified replicases described above as follows: (a) complete dependence on added RNA; (8) competence for prolonged (more than 5 hr) syn- thesis of RNA; (ce) ability to synthesize many times the input template; (d) satura- tion at low levels of RNA (1 pg RNA per 40 ug protein); and (e) virtually exclusive requirement for homologous template under optimal ionic conditions. In view of the very different states of purity, it is difficult to interpret the differ- ences in the properties observed with the enzymes reported here as compared with those detected!® or isolated’-}2 by others. Activity independent of added RNA® 9 suggests that the purification has not achieved removal of contaminating RNA and, in any case, precludes examination of template specificity. August et al.!0-!? isolated an enzyme from EL. colc infected with a mutant of f2 which is stimulated by a variety of RNA species, including host ribosomal and sRNA. It is conceivable that the f2-replicase is nonspecific. However, the August preparation requires 20 wg of RNA for each yg of protein, whereas the enzymes described here are fully saturated at 0.025 ug per ug of enzyme protein. The authors point out! that the inordinately large amounts of RNA required may be due to the detectable con- 586 BIOCHEMISTRY: HARUNA AND SPIEGELMAN Proc. N. A. 8S. tamination of their preparation with ribonuclease. Under the circumstances, one should withhold judgment on the significance of the apparent lack of specificity since it is open as yet to a rather trivial explanation. (3) Implications of template specificity requirement: The comparison of the two purified replicases reported establishes that each requires its homologous template. The experiment with the satellite-RNA (STNV) was a particularly interesting challenge. Reichmann" showed that the satellite virus contains only enough RNA to code for its own coat protein, which suggests that it, must employ the replicase of the companion virus (TNV) for its multiplication. This implies either that the satellite is related in sequence to the TNV virus, or that it possesses a feature per- mitting it to employ any viral RNA replicase. The fact that STNV-RNA did not serve as a template for either one of the two purified replicases implies that the answer will be found in at least partial sequence homology between STNV and TNV genomes, a prediction open to experimental test. The specificity relations exhibited raise the question of the mechanism used by the replicase to distinguish its template from other RNA molecules. The involve- ment of a beginning sequence is an obvious possibility. However, as will be shown,” the recognition mechanism is even more subtle, being designed to avoid replication of fragments of its own genome even if they contain the beginning sequence. It should be evident that the replicases are approaching a state of purity permitting the performance of unambiguous experiments which can hope to illu- minate the mechanism” of the RNA replicative process. Summary.—Two RNA replicases induced in the same host by unrelated RNA bacteriophages have been purified and their responses to various RNA molecules examined. Under optimal ionic conditions both are virtually inactive with heter- ologous RNA, including ribosomal and sRNA of the host. Neither replicase can funetion with the other’s RNA. Each recognizes the RNA genome of its origin and requires it as a template for synthetic activity. This discriminating selectivity of its replicase is of obvious advantage to a virus attempting to direct its own dupli- cation in a cellular environment replete with other RNA molecules. We should like to express our deep appreciation to Dr. I. Watanabe for making available to us the Qg virus which made possible the informative comparison reported. Similarly our profound thanks are due to Drs. W. Gilbert and J. D. Watson and to Miss Diane Vargo for providing the mutant Q-13. Finally, we are grateful to Mrs. Louise Jordan for skillful assistance in the per- formance of the experiments. * This investigation was supported by U.S. Public Health Service research grant CA-01094 from the National Cancer Institute and a grant from the National Science Foundation. ! Haruna, I., K. Nozu, Y. Ohtaka, and 8. Spiegelman, these ProcEEDINGS, 50, 905-911 (1963). 2 Doi, Roy H., and 8. Spiegelman, these PRocEEDINGS, 49, 353-360 (1963). 3 Watanabe, I., Nihon Rinsho, 22, 243 (1964). 4 Overby, L., G. H. Barlow, R. H. Doi, M. Jacob, and 8. Spiegelman, J. Bacterzol., in press. 5 Gesteland, R. F., Federation Proc., 24, 293 (1965). (Q-13 is a derivative of A-19, a RNase negative mutant reported here.) 6 Bresler, A. E., Biochim. Biophys. Acta, 61, 29-33 (1962). 7 August, J. T., P. J. Artiz, and J. Hurwitz, J. Biol. Chem., 237, 3786-3793 (1962). 8 Weissmann, C., P. Borst, R. H. Burdon, M. A. Billeter, and 8. Ochoa, these PRocEEDINGS, 51, 682 (1964). 9 Weissmann, C., L. Simon, and 8. Ochoa, these ProceEptnas, 49, 407-414 (1963). Vou. 54, L965 BIOCHEMISTRY: HARUNA AND SPIEGELMAN 587 " August, J. T., 8S. Cooper, L. Shapiro, and N. D. Zinder, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 99. “August, J. T., L. Shapiro, and L. Eoyang, J. Mol. Biol., 11, 257-271 (1965). “Shapiro, L., and J. T. August, J. Mol. Biol., 11, 272-284 (1965). 8 Krakow, J. S., and 8. Ochoa, these ProcEEDINGs, 49, 88-94 (1963). “ Nakamoto, T., and 8. B. Weiss, these PRocEEDINGS, 48, 880-887 (1962). ’ Baltimore, D., and R. M. Franklin, Biochem. Biophys. Res. Commun., 9, 388-392 (1963). 6 Reichmann, M. E., these ProceEDINGs, 52, 1009-1017 (1964). " Haruna, I., and 8. Spiegelman, manuscript in preparation.