A SEARCH FOR AN INTERMEDIATE INVOLVING A COMPLEMENTDURING SYNCHRONOUS SYNTHESIS BY A

PURIFIED RNA REPLICASE*

BY I. HARUNA AND S. SPIEGELMAN

DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS, URBANA

Communicated March 24, 1966

We have previously reported" 2 the isolation of two RNA replicases (RNA-dependent RNA polymerases) induced by serologically distinct3' 4 RNA bacterio-phages (MS-2 and Q$). Each enzyme exhibits a mandatory requirement foradded RNA and a unique preference for intact homologous RNA.The Q,8-replicase and the reaction it mediates possess the following features:

(1) freedom from detectable levels of RNase, RNA-phosphorylase, and DNA-dependent RNA polymerase; (2) capability of extensive synthesis and the genera-tion of a polynucleotide of the same base composition (Table 1, below) and molecu-lar weight5 as viral RNA; (3) a product which is completely effective in serving as atemplate;5' 6 (4) a product which is biologically active, being as competent as theoriginal RNA to program the synthesis of complete virus particles in protoplasts.6The properties summarized establish that our enzyme preparation is, in fact, pro-

ducing identical replicas of the templates. With the fulfillment of this necessaryprerequisite for the unambiguous analysis of the relevant replicating process, wefelt that a study of the mechanism could be safely initiated.

Since the reaction starts with viral RNA and ends with more of the same, everynecessary intervening stage must be represented in the reaction mixture. To pro-vide optimal conditions for the detection of intermediate forms, attention wasfocused on the early events, prior to the appearance of mature strands. Since Adoes not equal U in Qf3-RNA, complementary strands can be detected by base com-position. The present paper describes experiments pertinent to the following threequestions: (1) What is the initial base composition and how does it progress tothat of the mature viral RNA? (2) What is the degree of complementarity be-tween the early products and the template; in particular, does this exceed the self-complementarity of the viral RNA? (3) Is there evidence for a ribonuclease-re-sistant hydrogen-bonded intermediate between the early products and the template?

It will be recognized that experiments based on these questions are designed totest the validity of the widely accepted "double-stranded" mechanism7 of RNAreplication. Unfortunately, the data obtained do not confirm the straightforwardpredictions made by this model. The behavior of the base composition andcomplementarity do not support the conclusion that complements of the viral RNAare the initial products. Neither can any evidence be found for the appearance of aheat-denaturable RNase-resistant complex between early product and template.We are forced to the conclusion that other mechanisms ofRNA replication must stillbe entertained.Methods.-(1) Virus, enzyme, and assay conditions: The methods of preparing4 E. coli-Q-138

infected with Q3-virus,3 purification of the replicase, synthesis of radioactive substrates, assay forenzyme activity under standard conditions, and liquid scintillation counting on membrane filtershave been detailed previously." 2

(2) Removal of labeled riboside triphosphates from the reaction mixtures and purification of product:1256

VOL. 55, 1966 BIOCHEMISTRY: HARUNA AND SPIEGELMAN 1257

Since many of the experiments in the present investigation required the use of P32-riboside tri-phosphates of very high specific activity, it was necessary to remove these from the reaction mix-ture before the properties of the P32-product could be examined. Separation of product from thesubstrates was accomplished in the following manner: The reaction was stopped by adding EDTAto a final concentration of 0.01 M and 0.45 ml passed through a Sephadex G-25 column (0.8 by40 cm). Elution was carried out with SSC (0.15 M NaCl, 0.015 M Na citrate, pH 7.0). Figure 1shows a typical separation of H3-template and P32-product from P32-UTP. The labeled RNA'sare located as acid (10% TCA) precipitable cpm, and the UTP32 is found by spotting and drying20 X aliquots on membrane filters. The relevant fractions are pooled.RNase resistance can be carried out directly on the combined fractions containing P32-product

and HI-template. When annealing experiments are required, it is necessary to remove protein.Sodium dodecyl sulfate (SDS) is added to a final concentration of 0.4%. After 10 min at roomtemperature, the samples are extracted with phenol as described previously.2 The phenol is thenremoved with ether extractions and the product precipitated with alcohol. In our reactions themeasured RNase resistance of the product does not change as the result of the phenol-alcoholpurification, contrary to the experience of Borst and Weissmann.9

(3) Base composition: In addition to standard components,2 reaction mixtures for base com-position contain P32-ATP, P32-UTP, P32-GTP, and P32-CTP, each at a specific activity of 2 X107 cpm per gmole. After incubation for the indicated interval at 350C, the reactions are cooled,and 0.2 vol of a "washing mixture" (saturated pyrophosphate, saturated orthophosphate, 1 NHCl, and water in a ratio of 1:1:0.2:0.8) is added. To this are added 3 mg of E. coli bulk RNAand an equal volume of 10% perchloric acid (PCA). The resulting precipitate is then washed5 times with cold 5% PCA. The final precipitate is dissolved in 1 ml of 0.3 N NaOH and in-cubated at 370 for 18 hr. The nucleotides are then separated and analyzed for counts and opticaldensity on Dowex-1-formate, as detailed by Hayashi and Spiegelman.10

(4) Assay for ribonuclease resistance: Determination of RNase resistance can be carried out onaliquots from the Sephadex column or subsequent to the phenol alcohol purification with identicalresults. An aliquot dissolved in one ml of SSC containing 20 -y of RNase A at 30° for 90 min.When RNase T, was included, its concentration was also at 20 -y per ml. It was found by pre-liminary experiments that 90 min was more than adequate to yield stable plateaus of resistance.The reaction is stopped by adding 0.5 ml of the "washing mixture," 100 -y of DNA, and 0.3 ml of60% TCA. The sample is then washed on membrane filters several times with 10% TCA, asdetailed previously.', 2

(5) Annealing conditions: To permit comparison, the annealing conditions are similar to thoseused by Weissmann."1 The product is carried through the phenol-alcohol purification of § (2)of Methods and dissolved in 0.1 SSC. An aliquot (usually 0.05-0.08 ml in small serological tubes)plus the indicated amount of unlabeled Qfi-RNA is taken down to dryness in 2.5 hr in a vacuumoven at 550. Such samples usually contained between 700 and 1100 cpm (0.006-0.009 ,ug of p32.labeled product). The dried samples are then dissolved in 0.05 ml of 2 X SS, sealed, and keptat 600 for 5 hr for the annealing process. The contents of the tubes are quantitatively transferredwith 1.95 ml of SSC, and the RNase resistance assayed as described in § (4). Zero time samplesare dissolved in 2 ml of SSC without drying or annealing, for assay of initial RNase resistance.The drying step above is sufficient to yield approximately 60% of the hybrids which will eventuallybe obtained. This is not surprising in view of the exposure to high salt and concentration of thecomponents as the drying progresses.

Results.-(1) Base composition during early synthesis: QB-RNA possesses anadenine-to-uracil ratio of 0.75 (A/U of 1.33), permitting detection of comple-mentary strands, should they occur as the first step.

Table 1 summarizes the data obtained at various points in a synthesis extendingfrom 1.4 to 130 per cent of the input template, and Figure 2 describes the behaviorof the diagnostic A/U base ratio.The smooth progress of the base composition from a high A/U ratio to that

characteristic of Q#-RNA argues forcibly for a well-synchronized reaction. Sincethe ratio starts above 1.33, the curve must intersect this complementary value in

1258 BIOCHEMISTRY: HARUNA AND SPIEGELMAN PROC. N. A. S.

p32 2J X

6xlO5

5002,0|TMLT05<

1-5

9 P32-UTP A%/U OFCOMPPEMENT OFQEI RNA

4.105-

H~~~~~~~~~~~~~~~~~'101,5oo 3XIO5

H3TEMPLATE

A/U OF QII RNA

000DD 2KI05 \

soc- ,o1 5 P32PRODUCT

1 ~~~~~20 40 60 80 100 I20 140

5 6 7 1S9 101 % OF INPUT TEMPLATE SYNTHESIZEDFRACTION NUMBER FIG. 2.-A to U ratio during early

FIG. l.-Separation of term- periods of synchronized synthesis.plate and product RNA Details are given in Methods (2) andfrom UTP32. Each fraction (3) and the heading of Table 1, whichis 1 ml and the sample also contains complete information onvolume loaded is 0.45 ml. base composition.All other details as in §(2)of Methods.

order to reach the 0.75 ratio of the mature Q,3-RNA. There is, however, no evidenceof any tendency for the curve to linger at the complementary base composition.Further, as will be detailed elsewhere,'2 90 per cent of the RNA synthesized prior tothe 20 per cent point is less than the 28S value of Q13-RNA, a size which would alsopresumably characterize the complement.The data of Table 1 and Figure 2 clearly do not demand, or even encourage, an

interpretation in terms of complementary synthesis. They are completely explain-able by a synchronized appearance of identical partial copies of the template inwhich the first 100 nucleotides are comparatively rich in A and poor in U.

(2) Degree of self-complementarity of Qf3-RNA: Before considering experimentswhich propose to measure the complementarity of product to template (plusstrands), some attention must be paid to the logic of hybridization tests for com-plementary (minus) strands. Usually the labeled material is subjected to annealing

TABLE 1BASE COMPOSITION DURING SYNTHESIS OF Q,8-RNA

Reaction volume, Synthesis, %ml of template C A U G A/U10.0 1.4 22.0 36.3 19.4 22.2 1.875.0 2.3 23.1 32.4 22.6 21.6 1.432.0 11 23.4 28.2 24.7 23.5 1.141.0 37 24.6 27.0 26.6 21.9 1.010.50 60 24.0 26.0 28.3 21.7 0.920.25 130 25.3 23.9 29.7 21.1 0.81

Q16-RNA 24.7 22.3 29.4 23.7 0.76The numbers represent moles per cent. The details are as described in Methods. To provide adequate

quantities of synthesized product, the amounts of reaction used were adjusted according to the extent of syn-thesis permitted. In all cases, each ml of reaction contained 160 y of enzyme preparation and 4 y of Q#-RNA.Aliquots of 50 X were removed to determine the extent of synthesis by assay of acid-precipitable cpm. Theremainder was purified and analyzed for base composition as described in Methods. The values for Q#-RNAare from Overby et al.20

VOL. 55, 1966 BIOCHEMISTRY: HARUNA AND SPIEGELMAN 1259

10%- in the presence of excess unlabeled plus strands, sub-sequent to which a determination is made of labeled ri-

FRAGMENTED bonuclease-resistant RNA. It is important to emphasize5S.0;po-° ° FRAGMENTED that findingRNA which can hybridize with the plus strand

W < Tdoesnot warrant the conclusion that minus strands havebeen detected. The original template could possess

..45 ~ ~ 20 some self-complementarity; fragments of identical12345610 i20NgRA copies could then hybridize to their complementaryFIG. 3.-Assay of self- counterparts. Consequently, the unambiguous interpre-

complementarity of Q#-RNA.Qft-RNA, 0.02 -Y of either tation of annealing experiments demands a determinationi1tactc Q/ orNfragme5 ted of the self-complementarity of plus strands. Further,(12S) H3-Qj3-RNA (1.06 X the tests must be carried out under conditions similar105 cpm/-y) was annealedwith the indicated amounts to those employed in annealing with product.of unlabeled Q,8-RNA and Figure 3 shows the results of self-complementaritytested for RNase resistanceas in Methods. tests carried out with fragmented and intact H3-Qf#-

RNA annealed to increasing amounts of unlabeled Q,3-RNA. It is evident that 6 per cent of the H3-RNA can be converted to ribonucleaseresistant material by carrying it through an annealing test with an excess of identi-cal copies of itself. Note that, not unexpectedly, the use of fragments increases themeasured amount of self-complementarity. In addition, fragmented material iscloser to the situation expected in testing the products of a limited synthesis.The value of about 6 per cent provides, therefore, a lower limit of the extent of self-complementarity. Unless evidence can be provided that this number is exceeded, posi-tive results in annealing tests cannot be accepted as unequivocal evidence of the synthesisof negative strands.

(3) Assay of complementarity between early product and Q3-RNA: With thelower limit of self-complementarity established, it becomes possible to examine andinterpret complementarity between early product and template. Again, we focuson the period of synthesis in which most (>90%) of the newly synthesized materialis smaller than 28S.

Preliminary experiments indicated that a major proportion of the materialsynthesized in the first 5 per cent interval is hybridizable to the plus strand. Wehave already pointed out that complementarity in the initial interval of synthesisdoes not necessarily imply minus strand synthesis. This result can equally wellarise from complementarity between the beginning and some subsequent sequencein the plus strand, a possibility we proposed on other grounds.'3 It was necessary,therefore, to design an experiment that would reveal the degree of complementaritywhich obtains as the synthesis progresses through and beyond the first 5 per centinterval. Accordiiigly, a series of parallel syntheses were set up, each being labeledfor about a 5 per cent interval during different periods of synthesis. An aliquot ofeach was then challenged with 40 Sy (a fourfold excess) of Q,3-RNA in an annealingtest to determine the proportion of hybridizable product formed. Table 2 andFigure 4 record the complete details of an experiment of this nature.

It is evident that the resistance due to annealing is very high initially, and theextrapolation would indicate that it starts at close to 100 per cent. However, itdrops even before the first interval of synthesis is traversed so that only 77 per cent

1260 BIOCHEMISTRY: HARUNA AND SPIEGELMAN PROC. N. A. S.

TABLE 2ASSAY OF COMPLEMENTARITY BETWEEN PRODUCT AND TEMPLATE

Synthesis, --Recovery from:-_Labeling inter- Synthesis, % of Sephadex, Phenol- RNase Resistance of Annealing Test, %val, min cpm template % alcohol, % Before After Due to0-4 9,190 4.4 94.3 68.1 19.2 96.3 77.14-7 13,400 5.4 91.5 74.6 16.0 66.3 50.37-9 13,600 5.5 99.0 64.6 12.6 40.0 27.49-11 15,100 6.1 96.8 74.3 11.0 32.0 21.0

29.9* 18.8*11-13 15,900 6.5 96.9 70.8 10.1 28.8 18.7

29.2* 19.1*13-15 13,900 5.7 98.9 78.2 9.5 27.0 17.5

26.4* 16.9*To maximize synchrony, template and enzyme were incubated for 10 min in each case prior to addition of theriboside triphosphates. Each reaction volume was 0.50 ml and contained 34 y of enzyme and 1.6 y of RNA.This ratio of template to enzyme was just below the saturation value of this enzyme preparation to allow for maxi-mal participation of template strands. At the end of synthesis, 50-1i aliquots were removed for assay of P"2-RNAsynthesized. The remainder was purified through Sephadex and phenol and annealing carried out as detailed inMethods. In annealing, 0.005 -y of product was challenged with 40 y of cold Q#-RNA, which preliminary experi-ments indicated was 4 times the amount required for saturation of product. The starred duplicates (*) werechallenged with 20 -y of cold QjS-RNA.

5 lb 15 MINUTES of the whole first 4.4 per cent (corre-10 20 30 40 SYNTHESIS IN % OF sponding to about the first 120 nucle-I4.4 ~~~~~~TEMPLATE

_ 2- 9.8 otides) is complementary to some por-Z23 -E 15.3uonfE 4 2 1.4 tion of the plus strand. Note that 7724- _1121.4 pux 6 33.6 per cent of 4.4 per cent means that theW degree of complementarity of product

to the plus strand involves only 3.4o0 per cent of the viral RNA strand. TheIOO-'% ~FORMED\DURING ANNEALING complementarity continues to decrease,,, \\*throughout, falling to 17.5 per cent inZ R~the last interval examined, when theis . \ synthesis corresponds to 33 per cent*50 of the templates. The last threeZ \ points were challenged with both 20 y

and 40 -y plus strands. The fact thatBEFORE ANNEALING both yield the same values ensures that

, . , . the decreasing complementarity of the10 20 30 40 later products is not due to an insuffici-SYNTHESIS IN % OF TEMPLATE

ent supply of plus strands in the anneal-FIG. 4.-Complementarity of product to ing test.

template at various periods of synthesis. Com- Tplete numerical and experimental details are Thedataof Table2 and Figure 4 aregiven in Table 2, its legend, and in Methods. not easily reconcilable with the forma-The amount of resistance due to annealing isobtained by subtracting the resistance observed tion of complete complementaryprior to annealing from that measured after the strands as the first step in RNA rep-annealing. This is necessary since the initialRNase resistance of early product was shown to lication. On the other hand, they arebe insensitive to heat denaturation and due again readily explained by a mechanismprincipally to its high content of adenine. The * . * r. pupper portion gives the time plan of the six n whch ony replicas are produced andparallel syntheses, the hatched areas indicating the assumption that the beginning se-the interval of labeling with UTP"2 and the * * * anumbers representing synthesis finally achieved quence is rich i adenne and comple-in % of template. The last three samples were mentary to some other segment of thechallenged with 40 y (open circles) and 20 pr(closed cirdes) of unlabeled Q,6-RNA. plus strand rich i uracil.

VOL. 55, 1966 BIOCHEMISTRY: HARUNA AND SPIEGELMAN 1261

(4) Can a heat-denaturable ribonuclease-resistant structure be detected in the earlystages of RNA replication? We now turn our attention to the possible existence inour reaction mixtures of a "ribonuclease-resistant" structure involving a hydrogen-bonded duplex between plus and minus strands. This problem is forcibly raised bythe data in Table 2 and Figure 4. It will be noted that the early (e.g., 4.4%70)synthesis is much more resistant (19.4%) to RNase than is Q,8-RNA under the sameconditions (about 1%). However, the mere existence of RNase resistance cannotbe accepted as convincing evidence for a hydrogen-bonded duplex. The com-position might be such as to yield a relatively large resistant core, a possibility maderather likely by the high adenine content of the early products. One must, inaddition, examine the heat sensitivity of the resistant material. Unequivocaldecisions on the existence of hydrogen-bonded duplexes can be made only if suchsupplementary information is provided.

(a) Heat-sensitive ribonuclease-resistant structures in an abnormal replicasereaction: Before considering a normal replicating system, we prefer to begin bydescribing the results obtained with an abnormal reaction. This is meant to servetwo purposes. First, it demonstrates that when duplex structures are formed, theyare easily detected by the methods we employ. Second, it illustrates an avoidablesource of confusion generated by the presence of fragments or nucleases.We have previously shown'3 that when presented with fragments of its own ge-

nome, the replicase functions at less than 10 per cent of its capacity and ceases com-pletely in 30 min. This fragmentary reaction is not only brief but has little bio-logical future, since no complete molecules are ever produced. Table 3 summa-rizes typical data on the RNase resistance of the products formed. We see that64.8 per cent of the synthesized material is resistant to RNase A and 60.3 per cent tothe combined action of RNase A and T1. On heat denaturation, these values fallto 20.6 and 11.2 per cent, respectively. It is evident that under these conditions aconsiderable proportion of the product does enter into a resistant structure whichcan be destroyed by heat denaturation.

(b) Absence of heat-sensitive ribonuclease-resistant structures in normal replicasereactions: If one guarantees absence of nuclease activity in the replicase and in-tactness of template, no evidence can be detected of ribonuclease resistance whichis sensitive to heat destruction. Typical results are given in Table 4 which examinesRNase resistance of product and template at 5.3 and 11.0 per cent synthesis. TheH3 templates give their characteristically low resistance values and the P32 productsare clearly higher. However, these resistant cores are clearly not heat-sensitivestructures. Similar examinations at lower (0.6, 1.4, and 2.9%) and higher (57.5 and96.5%) levels of synthesis have yielded identical results.

TABLE 3RNAsE RESISTANCE OF PRODUCT SYNTHESIZED WITH FRAGMENTED TEMPLATES

Heat Time 0, ~ - Resistant Residue-denaturation RNase cpm Cpm Per cent

- A 584 378 64.8+ A 559 115 20.6- A + T, 584 332 60.3+ A + T1 559 62 11.2

In this case fragments of Q#-RNA isolated in a sucrose gradient in the 12S region were used. Theextent of synthesis corresponded to 1.7% of the template RNA. The resistance test was carried outin SSC for 90 min at 300C as described in Methods. Heat denaturation prior to resistance test was carried outfor 10 min at 1000C in SSC. A refers to pancreatic RNase and Ti to Tj RNase, isolated from Takadiastase(Sankyo Co., Ltd., Japan).

1262 BIOCHEMISTRY: HARUNA AND SPIEGELMAN PROC. N. A. S.

Discussion.-The "double-stranded" model of RNA replication postulates7 theintervention of a ribonuclease-resistant "replicating form" composed of one strandof viral RNA (the plus strand) complexed by hydrogen bonds to its complement(the minus strand). This duplex is presumed to serve as a template for the syn-thesis of plus strands analogous to the asymmetric transcription from the "RF-DNA" of 9X-174.14 15 Since the reaction being studied here requires the additionof plus strands as initiators, the model predicts that the first RNA to appear shouldbe of the minus variety and possess the following properties: (1) a base com-position complementary to the plus strand; (2) complete hybridizability to excessplus strands until the latter begin to appear in the reaction mixture; (3) a high in-itial resistance to RNase which is convertible to sensitivity by heat denaturation.The data obtained in the normal replicase reaction do not confirm any of these

predictions. Neither the progress of the base composition (Fig. 2) nor the ex-tent of complementarity between product and template (Fig. 4) offer acceptableevidence of an initial confinement to the synthesis of complete negative strands.Further, no denaturable ribonuclease-resistant structure could be detected eitherearly or later in the synthesis. The fact that such structures are easily observed inthe abbreviated abnormal reactions mediated by fragments lends added weight tothe negative findings with intact templates which produce biologically functionalRNA.

Recently, Weissmann9 and his collaborators arrived at the conclusion that theirearlier7 11, 16-18 evidence for a double-stranded intermediate was based on an artifactformed during RNA purification from crude enzyme preparations. We havenever observed such artifacts with RNA synthesized by purified replicase, eitherbefore or after phenol purification.The results of the normal in vitro replication are readily described by a mecha-

nism which involves the production of replicas without the intervention of an in-itially formed replicating duplex. The base composition, partial RNase resistance,and the limited complementarity of the early products are all explainable in termsof partial copies of an RNA strand possessing a beginning sequence rich in adenineand a complementary sequence rich in uracil near the end. This picture is in turnconsistent with the structure proposed'3 to explain how the replicase distinguishesboth sequence and intactness. Both decisions can be made in one act if the recog-

TABLE 4IRNASE RESISTANCE OF EARLY PRODUCT SYNTHESIZED WITH INTACT TEMPLATES

Heat de- - PP32_product-- _-_ H3-Template RNA--Synthesis, % natura- Time 0, --Residue- Time 0, -Residue --of template tion RNase cpm Cpm Per cent cpm Cpm Per cent

- A 663 87 13.0 1,861 31 1.7+ A 676 87 12.9 1,933 25 1.3

5.3- A + T1 663 42 6.3 1,861 12 0.6+ A + T, 676 40 5.9 1,933 10 0.5- A 1,802 206 11.4 2,418 43 1.8+ A 1,978 247 12.4 2,275 41 1.8

11.0- A + T, 1,802 126 7.0 2,418 18 0.7+ A + T, 1,978 123 6.7 2,275 15 0.7

Conditions are the same as in Table 3 except that intact templates were used.

VOL. 55, 1966 BIOCHEMISTRY: HARUNA AND SPIEGELMAN 1263

nition site on the template is a secondary structure formed by two complementarysequences, one at the beginning and the other at the end of the molecule.We emphasize again'9 that mechanisms which do not involve replicative duplexes

may still use the principle of complementarity via partial complements and otherdevices. However, other sorts of experiments are required to decide these issues.Summary.-If purified Qf3-replicase is presented with fragments of Q,3-RNA,

the reaction is slow (10% of normal), abbreviated (ceases within 30 min), and bio-logically abnormal. The product is small, biologically inactive, and much of it iscomplexed in a heat-denaturable RNase-resistant structure.In contrast, when activated by intact templates, Q,3-replicase produces virtually

unlimited amounts of complete and biologically competent replicas of Q8-RNA.Further, no heat-sensitive RNase-resistant material can be detected. Finally, neitherthe base composition nor annealability to "plus" strands revealed any compellingevidence for an initial appearance of complete complementary strands. All thedata on the early products are explainable in terms of partial copies of a templatepossessing a beginning sequence rich in adenine and another sequence comple-mentary to it further on in the chain.We recognize that negative evidence cannot logically be used to eliminate a con-

jectured mechanism. However, under conditions where convincing evidence mighthave been expected for the functioning of a double-stranded replicative intermedi-ate, none was forthcoming. Under the circumstances, we maintain at least for theQ,3-virus, that the double-stranded pathway has not been established and othermechanisms of RNA replication must still be entertained.

* This investigation was supported by U. S. Public Health Service research grant no. CA-01094from the National Cancer Institute and grant no. GB-2169 from the National Science Foundation.

1 Haruna, I., K. Nozu, Y. Ohtaka, and S. Spiegelman, these PROCEEDINGS, 50, 905 (1963).2 Haruna, I., and S. Spiegelman, these PROCEEDINGS, 54, 579 (1965).3 Watanabe, I., Nihon Rinsho, 22, 243 (1964).4 Overby, L. R., G. H. Barlow, R. H. Doi, Monique Jacob, and S. Spiegelman, J. Bacteriol.,

91, 442 (1966).5 Haruna, I., and S. Spiegelman, Science, 150, 884 (1965).6 Spiegelman, S., I. Haruna, I. B. Holland, G. Beaudreau, and D. Mills, these PROCEEDINGS,

54, 919 (1965).7 Ochoa, S., C. Weissmann, P. Borst, R. H. Burdon, and M. A. Billeter, Federation Proc., 23,

1285 (1964).8 Gesteland, R. F., Federation Proc., 24, 293 (1965).9 Borst, P., and C. Weissmann, these PROCEEDINGS, 54, 982 (1965).

10 Hayashi, M., and S. Spiegelman, these PROCEEDINGS, 47, 1564 (1961).11 Weissmann, C., these PROCEEDINGS, 54, 202 (1965).12 Haruna, I., and S. Spiegelman, manuscript in preparation.13 Haruna, I., and S. Spiegelnan, these PROCEEDINGS, 54, 1189 (1965).14 Hayashi, M., M. N. Hayashi, and S. Spiegelman, these PROCEEDINGS, 50, 664 (1963).15 Ibid., 51, 351 (1964).16 Weissmann, C., P. Borst, R. H. Burdon, M. A. Billeter, and S. Ochoa, these PROCEEDINGS,

51, 890 (1964).17 Weissmann, C., and P. Borst, Science, 142, 1188 (1963).18 Weissmann, C., P. Borst, R. H. Burdon, M. A. Billeter, and S. Ochoa, these PROCEEDINGS,

51, 682 (1964).19 Spiegelman, S., and I. Haruna, these PROCEEDINGS, in press.20 Overby, L. R., G. H. Barlow, R. H. Doi, Monique Jacob, and S. Spiegelman, J. Bacteriol.,

in press.