572. BIOCHEMISTRY: MORTLOCK ET AL. PROC. N. A. S.

5 Dische, Z., "Color reaction of nucleic acid components," in The Nucleic Acids, ed. E. Chargaffand J. Davidson (New York: Academic Press, 1955), Vol. 1, pp. 258-303.

6 Burton, K., Biochem. J., 62, 315 (1956).7Oyama, W. I., and H. Eagle, Proc. Soc. Exptl. Biol. Med., 91, 305 (1956).8 Marmur, J., J. Mol. Biol., 3, 208 (1961).9 Tamm, C., M. E. Hoder, and E. Chargaff, J. Bio. Chem., 195, 49 (1952).'0Szybalski, W., Anal. Biochem., 3, 267 (1962).11 Sueoka, N., J. Mol. Biol., 3, 31 (1961).12Trowne, P. W., and B. R. Rabin, these PROCEEDINGS, 52, 88 (1964).13Goldberg, I. H., M. Rabinowitz, and E. Reich, these PROCEEDINGS, 48, 2094 (1962).14 Siminoff, P., Appl. Microbiol., 9, 66 (1961).15 Crowther, D., and J. Melnick, Virology, 15, 65 (1964).I6 Smith, C. G., W. L. Lummis, and J. E. Grady, Cancer Res., 19, 847 (1959).17 Reich, E., Science, 143, 684 (1964).18 These experiments were performed by Drs. J. J. Vavra and H. E. Renis of The Upjohn

Company, and by Dr. J. J. Holland, University of California, Redwood, California.19 Liersch, M., and G. Hartman, Biochem. Z., 340, 390 (1964).20 DiMarco, A., et al., Nature, 201, 707 (1964).21 Ward, D., and E. Reich, Federation Proc., 24, 603 (1965).22 Chamberlin, M., and P. Berg, these PROCEEDINGS, 48, 81 (1962).23 Goldberg, I. H., Biochim. Biophys. Acta, 51, 201 (1961).24 Magee, W. E., Virology, 17, 604 (1962).

A BASIS FOR UTILIZATION OF UNNATURAL PENTOSESAND PENTJTOLS BY AEROBACTER AEROGENES*

BY R. P. MORTLOCK,t D. D. Fossirr, AND W. A. WOOD

DEPARTMENT OF BIOCHEMISTRY, MICHIGAN STATE UNIVERSITY, EAST LANSING, MICHIGAN

Communicated by H. G. Wood, June 18, 1966

Although not rigorously studied, it has long appeared that bacteria are unique intheir ability to metabolize those organic materials which are rarely, if ever, found innature. Aerobacter aerogenes, PRL R3, particularly illustrates this phenomenonwith its ability to utilize as a source of energy seven of the eight aldopentoses and allfour of the pentitols.' 2 Of this group of 11 structures, D-ribose, D-xylose, L-arabinose, D-arabitol, and ribitol are found in nature, whereas D-arabinose, D-lyxose, L-xylose, L-lyxose, xylitol, and L-arabitol3 seem to be rarely, if ever, en-countered in the natural environment.4 The routes of utilization of all of these C,compounds have been documented (Fig. 1).2. 5-18

Referring to Figure 1, it can be seen that metabolism of the unnatural compounds,D-lyxose and xylitol, for example, involves only one new or unique reaction foreach, i.e., isomerization of D-lyxose or oxidation of xylitol to D-xylulose. Thephosphorylation of D-xylulose is common to the route for utilization of D-xyloseand D-arabitol, both naturally occurring substrates readily attacked by A. aero-genes. Similarly, the conversions of L-xylose, L-lyxose, and L-arabitol involveeither isomerizations or an oxidation to L-xylulose.2 8 The subsequent reactions ofthe pathway are common to those for L-xylulose and L-ribulose2 19 which appear innature as metabolic intermediates in utilization of L-ascorbate20 and L-arabi-

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D-RIBOSE kinose D-RIBOSE-5-PHOSPHATE

D-ARABINOSEisomeroseisomerose

D-ARABINOSE - IsmrsD-RIBULOSE kinose-D-RIBULOSE D-RIBULOSE-5- PHOSPHATE

RIBITOL dehydrogenose i

D-XYLOSE isomerose 3-epimerose

D-LYXOSE isomerose

D-XYLULOSE kino, ID-XYLULOSE-5-PHOSPHATEI

D-ARABITOL dehydrogenose

XY LITOL dehydrogenose 4-epimerase

-ARABINOSE 4r- L-RIBULOSE k L-RIBULOSE-5-PHOSPHATE

L-XYLOSE -isomerose

3-epimeroseL-LYXOSE isomerose

L-XYLULOSE L-XYLULOSE-5-PHOSPHATE

L-ARAB TOL dehydrogenose IFIG. 1.-Pathways of pentose and pentitol dissimilation in A. aerogenes.

nose,'4 16, 18 respectively. Thus, only one unique reaction for each rare compoundis necessary for its dissimilation. The remainder of the sequence involves inducibleenzymes which function in dissimilation of naturally occurring pentoses and penti-tols and for which the necessary genes are pre-existent.2" 22

Thus, the central problem concerns the means by which the organism acquires anability to catalyze the unique isomerizations and dehydrogenations which convertthe rare structures to ketopentoses. From growth and induction experiments, ithas been established that the necessary enzymes for conversion of the naturallyoccurring structures to D-xylulose-5-phosphate are formed in response to inducer inthe usual way." 22 In addition, it is assumed that preservation of the genetic in-formation for these enzymes involves the usual selective forces. The same situationwould not be predicted for the unnatural substrates. The existence of genes direct-ing the synthesis of unique enzymes seems unlikely since an environment for theirselection presumably does not exist, and their eventual loss, if indeed they onceexisted, would result from mutations.Data indicating that a mutational event precedes the initiation of growth on the

unnatural substrates have been obtained" 22 and, hence, it is possible that thesemutations lead to the acquisition of a new enzyme. Yet this is unlikely becauseseveral specific acquisitions of a unique enzyme would be indicated from the abilityto grow on several of the unnatural substrates. Further, because of the relativelyshort period required to select a mutant population for many of these substrates,each mutation leading to a new enzyme would have to occur at a very high fre-quency. Thus, these acquisitions of a unique activity must be viewed as short-term

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events and not as evolutionary gain processes. Hence, the mutational acquisitionof unique enzymes seems unlikely.

It is the purpose of this report to present data which show, when considered withalready published observations, that a-fortuitous combination of circumstances canact as a mechanism which will allow growth on several unnatural pentoses andpentitols without necessitating the acquisition of a new enzyme.

Methods.-Aerobacter aerogenes was cultured at 300C on a salts-medium supple-mented with either 0.5 per cent carbohydrate, 1 per cent peptone, or casein hydroly-sate (Difco).' The method of Lin et al.23 was used to determine the proportion ofconstitutive cells and to isolate mutant strains, 4R1 and 5R1, after growth of thewild type on xylitol or L-arabitol, respectively. Preparation of extracts and anti-sera and methods of purification and determination of S20 values were described pre-viously.24 Pentitol dehydrogenases were assayed as the reduction of NAD bypentitol in the presence of semicarbazide buffer, pH 8.5,25 or by the oxidation ofNADH by ketopentose in tris buffer, pH 7.5.24 One unit of dehydrogenase cata-lyzed an absorbancy change of 1.0 per minute at 340 mu in a reaction volume of 0.15ml (1 = 1 cm). Isomerase activity was measured as the rate of ketopentose forma-tion by the method of Anderson and Wood.8 One unit of isomerase in 2.0 ml cata-lyzed the formation of 1 umole of ketopentose per hour at 370C. Protein was de-termined by the method of Lowry et al.,26 and ketopentose was estimated by thecysteine-carbozole test of Dische and Borenfreund.2YResults.-Enzymes for xylitol and L-arabitol utilization: The data of Mortlock

et al.24 28 show that the xylitol dehydrogenase activity of cells grown on xylitol isattributable to a ribitol dehydrogenase which is present in high levels. Both dehy-drogenase activities fractionate identically on DEAE cellulose and in classical pro-cedures; each has the same S20 value (6.0-6.2), and antibodies elicited by ribitoldehydrogenase purified from D-arabinose-grown cells inhibited xylitol dehydro-genase and ribitol dehydrogenase from xylitol-grown cells to the same extent (91-96%). Careful examination of extracts by DEAE-cellulose chromatography,sucrose density centrifugation, sucrose density electrophoresis, and by disk electro-phoresis failed to reveal the existence of another xylitol dehydrogenase.

Similarly, the L-arabitol dehydrogenase activity of cells grown on L-arabitol ap-pears to be attributable solely to ribitol dehydrogenase. The ratios of ribitol dehy-drogenase to L-arabitol dehydrogenase in the crude extract and various purifiedfractions were identical. Also, no other L-arabitol dehydrogenase was found ineffluents from DEAE cellulose columns or by disk electrophoresis.We have now crystallized (a) the inducible ribitol dehydrogenase from ribitol-

grown cells, (b) the ribitol dehydrogenase from a constitutive mutant isolated aftergrowth on xylitol, and (c) the ribitol dehydrogenase isolated from a constitutivemutant isolated after growth on L-arabitol.29 Each catalyzes the following reac-tions:

Ribitol + DPN+ >r± D-ribulose + DPNH + H+Xylitol + DPN+ T± D-xylulose + DPNH + H+L-arabitol + DPN+ 2± L-xylulose + DPNH + H+

As shown in Table 1, the specific activities of the crystalline material were identicalin each case (32,000). The relative maximum velocities for xylitol and L-arabitol

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TABLE ICOMPARISON OF RIBITOL D)EHYDROGENASE FROM DIFFERENT SOURCES

DehydrogenasecInducible, Constitutive mutant, Constitutive mutant,

ribitol culture xylitol culture L-arabitol cultureKM, ribitol 2.6 X 10-' 3.5 X 10-3 2.6 X 10-3

L-arabitol 2.9 X 10-1 2.5 X 10-1 4.0 X 10-1Xylitol 2.9 X 10-1 2.7 X 10-1 4.2 X 10-1

Ratio-V.., ribitol 100 100 100L-arabitol 35 39 46Xylitol 34 23 44

Heat sensitivity 4.5-4.8 5.0 5.0S20,. 6.0 5.8-6.2Gel electrophoresis

Protein (Rf) 0.40 0.4-0.41 0.39Activity (Rf) 0.40 0.40 0.40

Specific activity 32,000 30,000-32,000 32,000

determined from 1/s versus 1/v, s/v versus s, and v/s versus v calculations30 were23-46 per cent of the value for ribitol. The Km values for ribitol were essentiallyequal in each case at 3 X 10-3 M. Also, the values for xylitol and L-arabitol wereessentially identical, but approximately 100-fold greater than that of ribitol. Theheat sensitivities, determined at 56°C, were also virtually identical. Each dehy-drogenase migrated identically in polyacrylamide gel with a single protein bandwhich coincided with the band produced in a dye-coupled test for activity.31

Selection of constitutive mutants by growth on xylitol and L-arabitol: After growthfor 8 generations on xylitol, dehydrogenases for both ribitol and D-arabitol werepresent, as noted previously.2 Following transfer to peptone medium, the levels ofribitol dehydrogenase and D-arabitol dehydrogenase were followed in subsequentgenerations. As shown in Figure 2, ribitol dehydrogenase persists much longer thanexpected from an inducible enzyme deprived of inducer, whereas D-arabitol dehy-drogenase approximately follows the curve expected for dilution of a constantamount of enzyme during exponential growth in the absence of inducer. Thus,there appears to be a partial selection of constitutive mutants for ribitol dehydro-genase which are, at the same time, inducible for D-arabitol dehydrogenase. Induc-tion of D-arabitol dehydrogenase is considered to result from the presence of D-xylulose which arises from xylitol oxidation.2A mutant constitutive for ribitol dehydrogenase was then isolated after growth on

xylitol and subcultured sequentially on media containing (a) casein hydrolysate, (b)

100 FIG. 2.-Persistence of ribitol dehydro->_ |\genase in xylitol-grown cells after transfer to

80 t \ peptone medium. A. aerogenes,PRL R3, wasP grown for 8 generations in a xylitol-salts< t \ medium and used as inoculum for a peptone-O IRIBITOL DEHYDROGENASE salts medium. Samples were removed at timeE 60 intervals, cells harvested by centrifugation,o

^ washed, broken by sonic oscillation, and thecell-free extract was assayed for ribitol dehy-

40 drogenase. After growth on peptone, the perZ cent of the original activity remaining waso 2 \ ITHEORETICAL DILUTION plotted against the number of doublings of cello 20 \ 7 mass which had occurred. Ribitol dehydro-

D-ARABITOL DEHYDROGEN genase activity was assayed as the reductionof ribulose by NADH. Under the conditions

I employed (low D-xylulose level), D-xylulose0 4 8 12 16 20 24 reduction measured almost exclusively D-

GENERATIONS (FOLD DRY WEIGHT) arabitol dehydrogenase activity.

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TABLE 2RIBITOL DEHYDROGENASE CONTENT OF A CONSTITUTIVE MUTANT AFTER

GROWTH ON SEVERAL ENERGY SOURCESCumulative Generations by: Ribitol dehydrogenase*

Energy source Dry weight Viable count (units/mg protein)

Casein hydrolysate 0 0 5072.1 0.2 2424.2 1.86 15(7.0 4.23 1759.0 1889.7 19.8 23016.5 29.9 2342)5.5 39.6 27634.9 339

1)-Glucose 44.1 45Casein hydrolysate 53.3 464Xylitol 60.1 490

* Measured as D-ribulose reduction.A mutant strain 4R1 was isolated by plating a xylitol-grown culture and spraying the colonies as de-

scribed by Lin et al.23 A pink colony was picked and grown for 9 generations on xylitol-salts medium.The first casein hydrolysate medium was then inoculated. Then glucose-salts medium, casein hydroly-sate medium, and xylitol-salts medium were inoculated at the generation noted in the table. The cellswere then harvested, washed, ruptured by sonic oscillation, and assayed for ribitol dehydrogenase.Another portion was centrifuged, washed, and weighed, whereas a third portion was appropriately di-luted and plated on glucose-salts agar.

glucose, (c) casein hydrolysate, and (d) xylitol as energy sources (Table 2). After54 generations in the absence of inducer during which 9 generations were reared onglucose, the ribitol dehydrogenase level was 87 per cent of the initial value. Therewas, however, a slow decline in activity during growth on casein hydrolysate whichwas followed by a gradual increase. This constantly observed phenomenon may bedue to the presence of a small amount of catabolite repressor in the casein hydroly-sate.

In other experiments, after growth on xylitol or L-arabitol, cultures were platedon peptone agar and the number of mutants constitutive for ribitol dehydrogenasedetermined according to Lin et al.23 (Table 3). Over a period of 69 hr on xylitol,there was a rapid rise in the number of constitutive mutants until the populationwas almost entirely composed of constitutive mutants. Similarly, after eight gen-erations on L-arabitol, 332 of the 819 colonies observed, or about 41 per cent, wereconstitutive for ribitol dehydrogenase. After serial transfer on L-arabitol medium,the proportion of constitutive mutants approached 100 per cent.The isolated constitutive mutants, when inoculated into the standard xylitol or

L-arabitol medium, did not exhibit the lag of 2-4 days which characteristically pre-cedes growth of the wild type on L-arabitol or xylitol,' but grew immediately, as istypical of growth on a readily utilized substrate. Mutants isolated after growth onL-arabitol grew immediately on xylitol, and vice versa.Examination of cultures grown under conditions of ribitol dehydrogenase induc-

TABLE 3SELE(CTlrIoN OF CONsrrIITT1vE MUTANTS DURING GROWTH ON XYIITOL

Hours after --Cells per Mlinoculation Viable total Constitutive % Constitutive

0) 1.2 X 1() 0 020 1.7 X 1i)7 5.0( X 10 0:.342 9.5 X 1)7 1.9 X 1(7 19.569 3.7 X 10O' 3.7 X 109 100.0

Wild type A. aerogenes, PRL R3, was grown on D-glucose-salts medium and then transferred toxylitol-salts medium and incubated anaerobically at 30°C. Aliquots were plated at intervals onglucose-salts agar (total viable count) and on the constitutive assay medium of Lin et al.23

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TABLE 4RIBITOL DEHYDROGENASE ACTIVITIES OF CELL-FREE EXTRACTS OF

Aerobacter aerogenesRibitol dehydrogenase

Strain Energy source (units/mg protein) *Wild type Peptone 0-1Wild type Ribitol 46-154Wild type Xylitol 270-80()Wild type L-Arabitol 47-200Constitutive mutant, xylitol-grown (4R1) Peptone 750-100()Constitutive mutant, L-arabitol-grown (5R1) Peptone 650-850

* The spectrophotometric assay followed the oxidation of NADH in the presence of D-ribulose.' Oneunit of ribitol dehydrogenase catalyzes an absorbancy change of 1.0 per minute at 340 my in an assay volumeof 0.2 ml.

tion (ribitol-salts medium) failed to reveal the production of any constitutivemutants.Mutants isolated from typical pink colonies (Lin et al.23) and the wild type were

grown in the presence and absence of inducer (ribitol), and extracts were preparedby sonic treatment of washed suspensions. Table 4 summarizes the specific activi-ties of ribitol dehydrogenase in the wild type and these mutants. It can be seenthat the wild type was devoid of ribitol dehydrogenase when grown on peptone andthat ribitol dehydrogenase of moderate specific activity was induced during growthon ribitol. A considerably higher level of ribitol dehydrogenase was present aftergrowth on xylitol. In contrast, the constitutive mutants isolated after growth onxylitol or L-arabitol and then grown on peptone (without ribitol) had about 10-foldthe activity of the wild type grown on ribitol.

L-Xylose utilization: L-Xylose utilization has been shown to start with theisomerization of L-xylose to L-xylulose.8 The isomerase appears only after a lag ofabout 400 hr22 and is not elaborated under nonproliferating conditions. Aftergrowth on L-xylose, however, extracts contained high levels of D-arabinose iso-merase. As shown in Table 5, D-arabinose isomerase was present after growth onD-arabinose, but at least 10-fold more was present after growth on L-xylose. L-Xylose isomerase activity was barely detectable after growth on D-arabinose, butwas more than 10-fold higher after growth on L-xylose. The ratios of D-arabinoseisomerase to L-xylose isomerase activities remained constant whether D-arabinoseor L-xylose was used as an energy source.

After growth on L-xylose, the organism possessed specific activities for D-ara-binose isomerase of 239 and for L-xylose isomerase of 21. After 6.3 generations onpeptone-salts, the specific activities were 139 for D-arabinose isomerase and 6.6 forL-xylose isomerase. After 18 generations,a specific activity of 5.3 was still detected TABLE 5for D-arabinose isomerase, and after 25 ISOMERASE ACTIVITIES OF CELL-FREEgenerations, the value was 2.1. These EXTRACTS OF Aerobacter aerogenes

D-Arabinose L-Xylosevalues are considerably above those ex- isomerase isomeraseGrowth (units/mg (units/mgpected for an inducible enzyme undergoing substrate protein) protein)

dilution due to continued growth after re- Peptone 0 0D-Arabinose 17-39 0.5-1 .0moval of inducer and indicate the presence L-Xylose 239-584 8-15

of a high proportion of D-arabinose iso- After aerobic growth on a salts-medium con-merase-constitutivemutants. taining either 0.5% of D-arabinose, L-xylose, ormerase-constitutive ~~~~~1% peptone, the cells were harvested, washed,

Partial purification of D-arabinose iso- and extracts prepared with the French pressurems3ldofD-arabinoseratio of cell. Isomerase activity was determined by

merase (30-fold) did not change the ratio of measurement of ketopentose formed after incu-btoofaldopentose and enzyme at 370C.

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D-arabinose isomerase to L-xylose isomerase, and the configurational similarity ofthese two sugars about carbons 2 and 3 lends support to the idea that both isomer-izations are catalyzed by the same enzyme.Discussion.-For at least two and probably three of the rarely encountered C6

substrates (L-arabitol, xylitol, and L-xylose), growth is facilitated by a combinationof circumstances which circumvent a requirement for pre-existent genetic informa-tion (in the usual sense) for the production of enzymes specifically required for thedissimilation of these carbohydrates. The mechanism also circumvents thenecessity of acquiring de novo a unique enzyme by mutation. The organism doespossess genetic information for synthesis of a dehydrogenase and an isomerase whichcan utilize unnatural pentitols and pentoses related to the normal substrates. How-ever, this capacity at enzyme levels normally induced is insufficient to permit growthon the unnatural substrate. When mutation results in derepression and constitu-tive synthesis of high levels of these enzymes, utilization of the uncommon C5 com-pounds occurs at a sufficient rate to permit growth, and the mutant predominates inthe culture which emerges.An apparent inconsistency in this conclusion with respect to L-xylose utilization

is the fact that D-arabinose is rare in nature and that growth on D-arabinose ischaracteristic of the process of selection of mutants (i.e., growth is delayed in thefirst transfer and rapid thereafter).' Camyre and Mortlock32 have recently clarifiedthis situation by showing that both the L-xylose isomerase and D-arabinose iso-merase activities are due to an L-fuculose isomerase. All of the activities are induci-ble by L-fuculose, and the affinity for L-fuculose is higher than for D-arabinose orL-xylose. Since growth pccurs on L-fucose without a lag and L-fucose is found innature, growth on both L-xylose and D-arabinose may now be explained by thephenomenon just described.

Following our preliminary report of this phenomenon,28 Lerner et al.,3 publishedsimilar information for xylitol utilization by another strain of A. aerogenes andpointed to a possible extension of this phenomenon to evolutionary changes. How-ever widely applicable this phenomenon may be, it is certain that such circumstancesdo not govern growth on the unnatural pentose, D-lyxose, and the production of D-lyxose isomerase. Anderson and Allison34 have shown that D-lyxose isomerase is adistinct isomerase induced only by D-lyxose. Serial transfer on D-lyxose did notselect mutants constitutiye for D-lyxose isomerase. D-Mannose, a secondary sub-strate, did not induce the formation of D-lyxose isomerase, and the Km for D-man-nose was much higher than that for D-lyxose.

* Supported by a grant from the National Science Foundation. Contribution no. 3635 of theMichigan Agricultural Experiment Station.

t Postdoctoral fellow, National Institutes of Health. Present address: Department of Micro-biology, University of Massachusetts, Amherst, Mass.

1 Mortlock, R. P., and W. A. Wood, J. Bacteriol., 88, 838 (1964).2 Fossitt, D., R. P. Mortlock, R. L. Anderson, and W. A. Wood, J. Biol. Chem., 239, 2110

(1964).8 McCormick, D. B., and 0. Touster, Biochim. Biophys. Acts, 54, 598 (1961).4 Sowden, J. C., in The Carbohydrates, ed. Ward Pigman (1957), p. 77.6 Cohen, S. S., J. Biol. Chem., 201, 71 (1953).6 Stumpf, P. K., and B. L. Horecker, J. Biol. Chem., 218, 753 (1956).7 Burma, D. R., and B. L. Horecker, J. Biol. Chem., 231, 1053 (1958).8 Anderson, R. L., and W. A. Wood, J. Biol. Chem., 237, 296 (1962).

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9Ibid., 1029.'0Bhuyan, B. K., and F. J. Simpson, Can. J. Microbiol., 8, 737 (1962)." Fromm, H. J., J. Biol. Chem., 233, 1049 (1958).12Wood, W. A., M. J. McDonough, and L. B. Jacobs, J. Biol. Chem., 236, 2190 (1963).13 Hulley, S. B., S. E. Jorgensen, and E. C. C. Lin, Biochim. Biophys. Acta, 67, 219 (1963).4 Lampen, J. O., Abstracts, National Meeting of American Chemical Society, 1954, p. 44c.1 Simpson, F. J., M. J. Wolin, and W. A. Wood, J. Biol. Chem., 230, 457 (1958).16 Simpson, F. J., and W. A. Wood, J. Biol. Chem., 230, 473 (1958).7 Neish, A. C., and F. J. Simpson, Can. J. Biochem. Physiol., 32, 147 (1954).18 Altermatt, H. A., F. J. Simpson, and A. C. Neish, Can. J. Biochem. Physiol., 33, 615 (1955).19 Wolin, M. J., F. J. Simpson, and W. A. Wood, J. Biol. Chem., 232, 559 (1958).20 Cabib, E., in Annual Review of Biochemistry, ed. J. M. Luck (1963), p. 346.21 Mortlock, R. P., and W. A. Wood, Bacteriol. Proc., 110 (1963).22 Mortlock, R. P., and W. A. Wood, J. Bacterial., 88, 845 (1964).23 Lin, E. C. C., S. A. Lerner, and S. E. Jorgensen, Biochim. Biophys. Acta, 60, 422 (1962).24 Mortlock, R. P., D. Fossitt, D. H. Petering, and W. A. Wood, J. Bacterial., 89, 129 (1965).n Bonnichsen, R. K., and H. Theorell, Scand. J. Clin. Lab. Invest., 3, 58 (1951).26Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265

(1951).27 Dische, Z., and E. Borenfreund, J. Biol. Chem., 192, 583 (1951).28 Mortlock, R. P., D. Fossitt, and W. A. Wood, Bacteriol. Proc., 95 (1964).29 Fossitt, D., R. P. Mortlock, and W. A. Wood, Bacteriol. Proc., 82 (1965).30 Dowd, J. E., and D. S. Riggs, J. Biol. Chem., 240, 863 (1965).31 Moore, R. O., and Claud A. Villee, Science, 142, 389 (1963).32 Camyre, K. P., and R. P. Mortlock, J. Bacteriol., in press.33 Lerner, S. A., T. T. Wu, and E. C. C. Lin, Science, 146, 1313 (1964).34 Anderson, R. L., and D. P. Allison, J. Biol. Chem., 240, 2367 (1965).

SPECIFIC TEMPLATE REQUIREMENTS OF RNA REPLICASES*

By I. HARUNA AND S. SPIEGELMANDEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS, URBANA

Communicated June 21, 1965

We have previously1 reported the isolation of an RNA-dependent RNA polym-erase (termed a "replicase" for brevity) from E. coli infected with the RNA bac-teriophage MS-2. The purified enzyme showed a mandatory requirement foradded RNA and, furthermore, exhibited a unique preference for its homologousRNA. Ribosomal and sRNA of the host could not substitute as a template andneither of these cellular RNA types showed any ability to interfere with the tem-plate function of the viral RNA.We pointed out1 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 ignoresthe mass of pre-existent cellular RNA, a guarantee is provided that replication isfocused 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 valuenot 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

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